Cytotoxic peptides and antibody drug conjugates thereof

ABSTRACT

The present invention is directed to cytotoxic pentapeptides, to antibody drug conjugates thereof, and to methods for using the same to treat cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefits of U.S. Provisional Application No.61/561,255 filed Nov. 17, 2011, and U.S. Provisional Application No.61/676,423 filed Jul. 27, 2012, which are both hereby incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“PC71886ASequenceListing_ST25.txt” created on Oct. 24, 2012 and having asize of 45 KB. The sequence listing contained in this .txt file is partof the specification and is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is directed to novel peptide-based compoundsuseful as payloads in antibody-drug-conjugates (ADC's), andpayload-linker compounds useful in connection with ADC's. The presentinvention further relates to compositions including the aforementionedpayloads, payload-linkers and ADC's, and methods for using thesepayloads, payload-linkers and ADC's, to treat pathological conditionsincluding cancer.

BACKGROUND

Conjugation of drugs to antibodies, either directly or via linkers,involves a consideration of a variety of factors, including the identityand location of the chemical group for conjugation of the drug, themechanism of drug release, the structural elements providing drugrelease, and the structural modification to the released free drug. Inaddition, if the drug is to be released after antibody internalization,the mechanism of drug release must be consonant with the intracellulartrafficking of the conjugate.

While a number of different drug classes have been tried for deliveryvia antibodies, only a few drug classes have proved efficacious asantibody drug conjugates, while having a suitable toxicity profile. Onesuch class is the auristatins, derivatives of the natural productdolastatin 10. Representative auristatins include(N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine) and(N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine). However,there remains a need for additional auristatins with improvedproperties.

SUMMARY

The present invention relates to cytotoxic pentapeptides and antibodydrug conjugates thereof represented by formula:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is hydrogen, C₁-C₈ alkyl, or C₁-C₈ haloalkyl, or R¹ is a linker or alinker-antibody such

as

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-or —(C₃-C₈ heterocyclo)-C₁-C₁₀alkylene-;

Z is

G is halogen, —OH, —SH or —S—C₁-C₆ alkyl;

L is an antibody;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are defined as either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl or            halogen; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are defined as either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

or R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

R⁶ is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl;

R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄ aryl;

R¹³ is C₁-C₁₀ heterocyclyl; and

R⁷ is F, Cl, I, Br, NO₂, CN and CF₃;

h is 1, 2, 3, 4 or 5; and

X is O or S;

provided that when R^(3A) is hydrogen X is S.

The present invention relates to cytotoxic pentapeptides and antibodydrug conjugates thereof represented by formula:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are defined as either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl, halogen            or hydrogen; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are defined as either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂,—CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl;

R¹¹ is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, or R¹¹ is a linker orlinker-antibody such as

Y is C₂-C₂₀ alkylene or C₂-C₂₀ heteroalkylene; C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z is

G is halogen, —OH, —SH or —S—C₁-C₆ alkyl; L is an antibody;

R⁷ is F, Cl, I, Br, NO₂, CN and CF₃,

h is 1, 2, 3, 4 or 5; and

X is O or S.

Another aspect of the invention relates to pharmaceutical compositionsincluding an effective amount of any one of the aforementioned compoundsand/or any one of the aforementioned antibody drug conjugates and apharmaceutically acceptable carrier or vehicle.

Another aspect of the invention relates to a method of using aneffective amount of any one of the aforementioned compounds and/or anyone of the aforementioned antibody drug conjugates to treat cancer byadministering to a patient in need thereof an effective amount of saidcompound and/or conjugate.

Another aspect of the invention relates to a method of treating cancerwherein said cancer includes a tumor, metastasis, or other disease ordisorder characterized by uncontrolled cell growth wherein said canceris selected from the group consisting of carcinomas of the bladder,breast, cervix, colon, gliomas, endometrium, kidney, lung, esophagus,ovary, prostate, pancreas, melanoma, stomach, and testes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a graph of anti-tumor activity of four conjugates (eachadministered at 1 mg/kg, Q4dx4) plotted as tumor volume over time.

FIG. 2 depicts a graph of anti-tumor activity of six conjugates (eachadministered at 1 mg/kg, Q4dx4) plotted as drug-treated tumorvolume/vehicle-treated tumor volume over time.

FIG. 3 depicts the results of the testing of H(C)-#D54 and H(C)-vcMMAEat 1 mg/kg s

FIGS. 4A, 4B and 4C depict [A] results of testing of H(C)-#D54 andH(K)-MCC-DM1 in a MDA-MB-361-DYT2 mouse xenograft in vivo screeningmodel; [B] results of the testing of H(C)-vcMMAE and H(C)-mcMMAF in aMDA-MB-361-DYT2 mouse xenograft in vivo screening model; and [α] acomparison of the calculated T/C for all four conjugates. Mice weretreated q4dx4, starting on day 1.

FIGS. 5A, 5B, 5C, 5D, 5E and 5F depict the dose response results of thetesting [A] H(C)-#D54, [B] H(C)-vcMMAE, [α] H(C)-mcMMAF and [D]H(K)-MCC-DM1 in a N87 mouse xenograft in vivo model; [E] a comparison ofH(C)-#D54 and H(C)-vcMMAE; and [F] a comparison of T/C for all fourconjugates. Mice were treated q4dx4, starting on day 1.

FIG. 6 depicts the dose response results of the testing H(C)-#A115 at 1mpk, 3 mpk and 10 mpk, in a N87 mouse xenograft in vivo model. Mice weretreated q4dx4, starting on day 1.

FIG. 7 shows data comparing humanized antibody hu08 conjugated tovc-0101 or mc-3377, tested in an in vivo xenograft model with PC3mM2cells, a human prostate cancer cell line that expresses the IL-13Rα2receptor.

FIGS. 8A through E show [A] the efficacy of rat-human chimericanti-Notch ADCs dosed at 5 mg/kg in HCC2429 lung xenografts; [B and C]the efficacy of rat-human chimeric anti-Notch ADCs dosed at 5 mg/kg inMDA-MB-468 breast xenografts; [D and E] the efficacy of rat-humanchimeric anti-Notch ADCs dosed at 5 mg/kg in N87 gastric xenograft.

DETAILED DESCRIPTION

The present invention is directed to cytotoxic pentapeptides, toantibody drug conjugates comprising said cytotoxic pentapeptides, and tomethods for using the same to treat cancer and other pathologicalconditions. The invention also relates to methods of using suchcompounds and/conjugates in vitro, in situ, and in vivo for thedetection, diagnosis or treatment of mammalian cells, or associatedpathological conditions.

DEFINITIONS AND ABBREVIATIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings. When trade names are usedherein, the trade name includes the product formulation, the genericdrug, and the active pharmaceutical ingredient(s) of the trade nameproduct, unless otherwise indicated by context.

The term “antibody” (or “Ab”) herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,monospecific antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments that exhibit the desired biologicalactivity. An intact antibody has primarily two regions: a variableregion and a constant region. The variable region binds to and interactswith a target antigen. The variable region includes a complementarydetermining region (CDR) that recognizes and binds to a specific bindingsite on a particular antigen. The constant region may be recognized byand interact with the immune system (see, e.g., Janeway et al., 2001,Immuno. Biology, 5th Ed., Garland Publishing, New York). An antibody canbe of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The antibody can bederived from any suitable species. In some embodiments, the antibody isof human or murine origin. An antibody can be, for example, human,humanized or chimeric.

The terms “specifically binds” and “specific binding” refer to antibodybinding to a predetermined antigen. Typically, the antibody binds withan affinity of at least about 1×10⁷ M⁻¹, and binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method.

The term “monoclonal antibodies” specifically includes “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical to or homologous with the corresponding sequence of antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical toor homologous with the corresponding sequences of antibodies derivedfrom another species or belonging to another antibody class or subclass,as well as fragments of such antibodies, so long as they exhibit thedesired biological activity.

As used herein, “H(C)-” refers to trastuzumab (trade name HERCEPTIN®)which is a monoclonal antibody that interferes with the HER2/neureceptor, bound through one of its' cystine to compound of theinvention. As used herein, “H(K)-” refers to trastuzumab which is amonoclonal antibody that interferes with the HER2/neu receptor, boundthrough one of its' lysines to compound of the invention.

An “intact antibody” is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H1), C_(H2), C_(H3) and C_(H4), as appropriate forthe antibody class. The constant domains may be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variants thereof.

An intact antibody may have one or more “effector functions”, whichrefers to those biological activities attributable to the Fc region(e.g., a native sequence Fc region or amino acid sequence variant Fcregion) of an antibody. Examples of antibody effector functions includecomplement dependent cytotoxicity, antibody-dependent cell-mediatedcytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis.

An “antibody fragment” comprises a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments, diabodies, triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, scFv, scFv-Fc, multispecific antibodyfragments formed from antibody fragment(s), a fragment(s) produced by aFab expression library, or an epitope-binding fragments of any of theabove which immuno specifically bind to a target antigen (e.g., a cancercell antigen, a viral antigen or a microbial antigen).

The term “variable” in the context of an antibody refers to certainportions of the variable domains of the antibody that differ extensivelyin sequence and are used in the binding and specificity of eachparticular antibody for its particular antigen. This variability isconcentrated in three segments called “hypervariable regions” in thelight chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FRs). The variable domains of native heavy and light chains eachcomprise four FRs connected by three hypervariable regions.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (L3) in the heavy chain variabledomain; Kabat et al. (Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (e.g.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (142) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, 1987, J. Mol. Biol.196:901-917). FR residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

A “single-chain Fv” or “scFv” antibody fragment comprises the V.sub.Hand V.sub.L domains of an antibody, wherein these domains are present ina single polypeptide chain. Typically, the Fv polypeptide furthercomprises a polypeptide linker between the V.sub.H and V.sub.L domainswhich enables the scFv to form the desired structure for antigenbinding. For a review of scFv, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabody” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 0 404 097; WO 93/11161; and Hollinger et al., 1993, Proc.Natl. Acad. Sci. USA 90:6444-6448.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., 1986, Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta,1992, Curr. Op. Struct. Biol. 2:593-596.

As used herein, “isolated” means separated from other components of (a)a natural source, such as a plant or animal cell or cell culture, or (b)a synthetic organic chemical reaction mixture.

As used herein, “purified” means that when isolated, the isolatecontains at least 95%, and in another aspect at least 98%, of a compound(e.g., a conjugate) by weight of the isolate.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is a tumor cell, e.g., a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering; and nuclear/chromatin condensationalong with DNA fragmentation can be evaluated by any increase inhypodiploid cells.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may inhibit the growth of and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic. For cancertherapy, efficacy can, for example, be measured by assessing the time todisease progression (TTP) and/or determining the response rate (RR).

The term “substantial amount” refers to a majority, i.e. greater than50% of a population, of a mixture or a sample.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC). The metabolic process or reaction may be an enzymaticprocess such as proteolytic cleavage of a peptide linker of the ADC.Intracellular metabolites include, but are not limited to, antibodiesand free drug which have undergone intracellular cleavage after entry,diffusion, uptake or transport into a cell.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on an ADC or the like,whereby the covalent attachment, e.g., the linker, between the drugmoiety and the antibody is broken, resulting in the free drug, or othermetabolite of the conjugate dissociated from the antibody inside thecell. The cleaved moieties of the ADC are thus intracellularmetabolites.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of a drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, a cytostatic oran anti-proliferative effect of a ADC or an intracellular metabolite ofsaid ADC. Cytotoxic activity may be expressed as the IC₅₀ value, whichis the concentration (molar or mass) per unit volume at which half thecells survive.

A “disorder” is any condition that would benefit from treatment with adrug or antibody-drug conjugate. This includes chronic and acutedisorders or diseases including those pathological conditions whichpredispose a mammal to the disorder in question. Non-limiting examplesof disorders to be treated herein include benign and malignant cancers;leukemia and lymphoid malignancies, neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, angiogenic and immunologicdisorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition or disorder in mammals that is typicallycharacterized by unregulated cell growth. A “tumor” comprises one ormore cancerous cells.

Examples of a “patient” include, but are not limited to, a human, rat,mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird andfowl. In an exemplary embodiment, the patient is a human.

The terms “treat” or “treatment,” unless otherwise indicated by context,refer to therapeutic treatment and prophylactic measures to preventrelapse, wherein the object is to inhibit or slow down (lessen) anundesired physiological change or disorder, such as the development orspread of cancer. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadyhaving the condition or disorder as well as those prone to have thecondition or disorder.

In the context of cancer, the term “treating” includes any or all ofinhibiting growth of tumor cells, cancer cells, or of a tumor;inhibiting replication of tumor cells or cancer cells, lessening ofoverall tumor burden or decreasing the number of cancerous cells, andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of inhibiting replication of cells associated with anautoimmune disease state including, but not limited to, cells thatproduce an autoimmune antibody, lessening the autoimmune-antibody burdenand ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includesany or all of: inhibiting the growth, multiplication or replication ofthe pathogen that causes the infectious disease and ameliorating one ormore symptoms of an infectious disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indication(s), usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

As used herein, the terms “cell,” “cell line,” and “cell culture” areused interchangeably and all such designations include progeny. Thewords “transformants” and “transformed cells” include the primarysubject cell and cultures or progeny derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

Unless otherwise indicated, the term “alkyl” by itself or as part ofanother term refers to a straight chain or branched, saturatedhydrocarbon having the indicated number of carbon atoms (e.g., “C₁-C₈”alkyl refer to an alkyl group having from 1 to 8 carbon atoms). When thenumber of carbon atoms is not indicated, the alkyl group has from 1 to 8carbon atoms. Representative straight chain C₁-C₈ alkyls include, butare not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,n-heptyl and n-octyl; while branched C₁-C₈ alkyls include, but are notlimited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl,and -2-methylbutyl; unsaturated C₂-C₈ alkyls include, but are notlimited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl and 3-methyl-1-butynyl.

Unless otherwise indicated, “alkylene,” by itself of as part of anotherterm, refers to a saturated, branched or straight chain or cyclichydrocarbon radical of the stated number of carbon atoms, typically 1-18carbon atoms, and having two monovalent radical centers derived by theremoval of two hydrogen atoms from the same or two different carbonatoms of a parent alkane. Typical alkylene radicals include, but are notlimited to: methylene (—CH₂—), 1,2-ethylene —CH₂CH₂—), 1,3-propylene(—CH₂CH₂CH₂—), 1,4-butylene (—CH₂CH₂CH₂CH₂—), and the like. A “C₁-C₁₀”straight chain alkylene is a straight chain, saturated hydrocarbon groupof the formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

Unless otherwise indicated, the term “heteroalkyl,” by itself or incombination with another term, means, unless otherwise stated, a stablestraight or branched chain hydrocarbon, or combinations thereof, fullysaturated or containing from 1 to 3 degrees of unsaturation, consistingof the stated number of carbon atoms and from one to three heteroatomsselected from the group consisting of O, N, Si and S, and wherein thenitrogen and sulfur atoms may optionally be oxidized and the nitrogenheteroatom may optionally be quaternized. The heteroatom(s) O, N and Smay be placed at any interior position of the heteroalkyl group. Theheteroatom Si may be placed at any position of the heteroalkyl group,including the position at which the alkyl group is attached to theremainder of the molecule. Up to two heteroatoms may be consecutive.

Unless otherwise indicated, the term “heteroalkylene” by itself or aspart of another substituent means a divalent group derived fromheteroalkyl (as discussed above). For heteroalkylene groups, heteroatomscan also occupy either or both of the chain termini.

Unless otherwise indicated, “aryl,” by itself or an part of anotherterm, means a substituted or unsubstituted monovalent carbocyclicaromatic hydrocarbon radical of 6-20, preferably 6-14, carbon atomsderived by the removal of one hydrogen atom from a single carbon atom ofa parent aromatic ring system. Typical aryl groups include, but are notlimited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like. A substitutedcarbocyclic aromatic group (e.g., an aryl group) can be substituted withone or more, preferably 1 to 5, of the following groups: C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected from—H, C₁-C₈ alkyl and unsubstituted aryl. In some embodiments, asubstituted carbocyclic aromatic group can further include one or moreof: —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′. “Arylene” is thecorresponding divalent moiety.

“Substituted alkyl” means an alkyl in which one or more hydrogen atomsare each independently replaced with a substituent. Typical substituentsinclude, but are not limited to, —X, —R, —O—, —OR, —SR, —S⁻, —NR₂, —NR₃,═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃,—NRC(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR,—S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO₃ ²⁻, PO₃H₂, —AsO₂H₂, —C(═O)R,—C(═O)X, —C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR, —C(═O)SR, —C(═S)SR,—C(═O)NR₂, —C(═S)NR₂, or —C(═NR)NR₂, where each X is independently ahalogen: —F, —Cl, —Br, or —I; and each R is independently —H, C₁-C₂₀alkyl, C₁-C₂₀ heteroalkyl, C₆-C₂₀ aryl, C₁-C₁₀ heterocyclyl, aprotecting group or a prodrug moiety. Aryl, alkylene and heteroalkylenegroups as described above may also be similarly substituted.

Unless otherwise indicated, “aralkyl” by itself or part of another term,means an alkyl group, as defined above, substituted with an aryl group,as defined above.

Unless otherwise indicated, “C₁-C₁₀ heterocyclyl” by itself or as partof another term, refers to a monovalent substituted or unsubstitutedaromatic or non-aromatic monocyclic, bicyclic or tricyclic ring systemhaving from 1 to 10, preferably 3 to 8, carbon atoms (also referred toas ring members) and one to four heteroatom ring members independentlyselected from N, O, P or S, and derived by removal of one hydrogen atomfrom a ring atom of a parent ring system. One or more N, C or S atoms inthe heterocyclyl can be oxidized. The ring that includes the heteroatomcan be aromatic or nonaromatic. Unless otherwise noted, the heterocyclylis attached to its pendant group at any heteroatom or carbon atom thatresults in a stable structure. Representative examples of a C₁-C₁₀heterocyclyl include, but are not limited to, tetrahyrofuranyl,oxetanyl, pyranyl, pyrrolidinyl, piperidinyl, piperazinyl, benzofuranyl,benzothiophene, benzothiazolyl, indolyl, benzopyrazolyl, pyrrolyl,thiophenyl (thiopene), furanyl, thiazolyl, imidazolyl, pyrazolyl,triazolyl, quinolinyl including moieties such as1,2,3,4-tetrshyhro-quinolinyl, pyrimidinyl, pyridinyl, pyridonyl,pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, tetrazolyl, epoxide,oxetane and BODIPY (substituted or unsubstituted). A C₁-C₁₀ heterocyclylcan be substituted with up to seven groups including, but not limitedto, C₁-C₈ alkyl, C₁-C₈ heteroalkyl, —OR′, aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —S(═O)₂R′,—S(O)R′, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′is independently selected from —H, C₁-C₈ alkyl, C₁-C₈ heteroalkyl andaryl. In some embodiments, a substituted heterocyclyl can also includeone or more of: —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′.“Heterocyclo” “C₁-C₁₀ heterocyclo” is the corresponding divalent moiety.

Unless otherwise indicated, “heteroaralkyl” by itself or part of anotherterm, means an alkyl group, as defined above, substituted with anaromatic heterocyclyl group, as defined above. Heteroaralklo is thecorresponding divalent moiety.

Unless otherwise indicated, “C₃-C₈ carbocyclyl” by itself or as part ofanother term, is a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent,substituted or unsubstituted, saturated or unsaturated non-aromaticmonocyclic or bicyclic carbocyclic ring derived by the removal of onehydrogen atom from a ring atom of a parent ring system. RepresentativeC₃-C₈ carbocyclyl include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl,1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctadienyl, bicyclo(1.1.1.)pentane, and bicyclo(2.2.2.)octane. AC₃-C₈ carbocyclyl group can be unsubstituted or substituted with up toseven groups including, but not limited to, C₁-C₈ alkyl, C₁-C₈heteroalkyl, —OR′, aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —S(═O)₂R′, —S(═O)R′, —OH, -halogen,—N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ is independentlyselected from —H, C₁-C₈ alkyl, C₁-C₈ heteroalkyl and aryl. “C₃-C₈carbocyclo” is the corresponding divalent moiety.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms, McGraw-HillBook Company, New York (1984); and Eliel and Wilen, Stereochemistry ofOrganic Compounds, John Wiley & Sons, Inc., New York (1994). Manyorganic compounds exist in optically active forms, i.e., they have theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L, or R and S, are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or 1 meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesestereoisomers are identical except that they are mirror images of oneanother. A specific stereoisomer may also be referred to as anenantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

An amino acid “derivative” includes an amino acid having substitutionsor modifications by covalent attachment of a parent amino acid, such as,e.g., by alkylation, glycosylation, acetylation, phosphorylation, andthe like. Further included within the definition of “derivative” is, forexample, one or more analogs of an amino acid with substituted linkages,as well as other modifications known in the art.

A “natural amino acid” refers to arginine, glutamine, phenylalanine,tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine,proline, glutamic acid, aspartic acid, threonine, cysteine, methionine,leucine, asparagine, isoleucine, and valine, unless otherwise indicatedby context.

“Protecting group” refers to a moiety that when attached to a reactivegroup in a molecule masks, reduces or prevents that reactivity. Examplesof protecting groups can be found in T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons,New York, 1999, and Harrison and Harrison et al., Compendium ofSynthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996),which are incorporated herein by reference in their entirety.Representative hydroxy protecting groups include acyl groups, benzyl andtrityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allylethers. Representative amino protecting groups include, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl(Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES),trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC),and the like.

Examples of a “hydroxyl protecting group” include, but are not limitedto, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranylether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether,triethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silylether, triphenylmethyl silyl ether, acetate ester, substituted acetateesters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, but are not limited to, a halide (e.g., chloride,bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound.The compound typically contains at least one amino group, andaccordingly acid addition salts can be formed with this amino group.Exemplary salts include, but are not limited to, sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, malate, gentisinate, fumarate, gluconate,glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a compound or conjugateof the invention. Examples of solvents that form pharmaceuticallyacceptable solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

The terms “loading” or “drug loading” or “payload loading” represent orrefer to the average number of payloads (“payload” and “payloads” areused interchangeable herein with “drug” and “drugs”) per antibody in anADC molecule. Drug loading may range from 1 to 20 drugs per antibody.This is sometimes referred to as the DAR, or drug to antibody ratio.Compositions of the ADCs described herein typically have DAR's of from1-20, and in certain embodiments from 1-8, from 2-8, from 2-6, from 2-5and from 2-4. Typical DAR values are 2, 4, 6 and 8. The average numberof drugs per antibody, or DAR value, may be characterized byconventional means such as UV/visible spectroscopy, mass spectrometry,ELISA assay, and HPLC. The quantitative DAR value may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADCs having a particular DAR value maybe achieved by means such as reverse phase HPLC or electrophoresis. DARmay be limited by the number of attachment sites on the antibody. Forexample, where the attachment is a cysteine thiol, an antibody may haveonly one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a Linker unitmay be attached. In some embodiments, the cysteine thiol is a thiolgroup of a cysteine residue that forms an interchain disulfide bond. Insome embodiments, the cysteine thiol is a thiol group of a cysteineresidue that does not form an interchain disulfide bond. Typically,fewer than the theoretical maximum of drug moieties are conjugated to anantibody during a conjugation reaction. An antibody may contain, forexample, many lysine residues that do not react with a linker or linkerintermediate. Only the most reactive lysine groups may react with areactive linker reagent.

Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which may be linked to a drug via a linker. Mostcysteine thiol residues in the antibodies exist as disulfide bridges andmust be reduced with a reducing agent such as dithiothreitol (DTT). Theantibody may be subjected to denaturing conditions to reveal reactivenucleophilic groups such as lysine or cysteine. The loading(drug/antibody ratio) of an ADC may be controlled in several differentmanners, including: (i) limiting the molar excess of drug-linkerrelative to the antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification. Where more than one nucleophilic groupreacts with a drug-linker then the resulting product is a mixture ofADC's with a distribution of one or more drugs moieties per antibody.The average number of drugs per antibody may be calculated from themixture by, for example, dual ELISA antibody assay, specific forantibody and specific for the drug. Individual ADC's may be identifiedin the mixture by mass spectroscopy, and separated by HPLC, e.g.,hydrophobic interaction chromatography.

Below is a list of abbreviations and definitions that may not otherwisebe defined or described in this application: DMSO (refers to dimethylsulfoxide), HRMS (refers to high resolution mass spectrometry), DAD(refers to diode array detection), TFA (refers to 2,2,2-trifluoroaceticacid or trifluoroacetic acid), TFF (refers to tangential flowfiltration), EtOH (refers to ethanol), MW (refers to molecular weight),HPLC (refers to high performance liquid chromatography), prep HPLC(refers to preparative high performance liquid chromatography), etc.(refers to and so forth), trityl (refers1,1′,1″-ethane-1,1,1-triyltribenzene), THF (refers to tetrahydrofuran),NHS (refers to 1-Hydroxy-2,5-pyrrolidinedione), Cbz (refers tocarboxybenzyl), eq. (refers to equivalent), n-BuLi (refers ton-butyllithium), OAc (refers to acetate), MeOH (refers to methanol),i-Pr (refers to isopropyl or propan-2-yl), NMM (refers to4-methylmorpholine), and “-” (in a table refers to no data available atthis time).

Compounds and Antibody Drug Conjugates Thereof

One aspect of the invention relates to a compound of formula I:

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein, independently for each occurrence,

W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen        or aralkyl; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl or            halogen; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

or R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

R⁶ is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, —C₂-C₈alkynyl or —C₁-C₈ haloalkyl;

R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄ aryl;

R¹³ is C₁-C₁₀ heterocyclyl; and

X is O or S;

provided that when R^(3A) is hydrogen X is S.

Another aspect of the invention relates to compound of formula IIa:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-; —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-or —(C₃-C₈ heterocyclo)-C₁-C₁₀alkylene-;

Z is

G is halogen, —OH, —SH or —S—C₁-C₆ alkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl or            halogen; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

or R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

R⁶ is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl;

R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄ aryl;

R¹³ is C₁-C₁₀ heterocyclyl; and

R⁷ is independently selected for each occurrence from the groupconsisting of F, Cl, I, Br, NO₂, CN and CF₃;

R¹⁰ is hydrogen, —C₁-C₁₀alkyl, —C₃-C₈-carbocyclyl, -aryl,—C₁-C₁₀heteroalkyl, —C₃-C₈heterocyclo, —C₁-C₁₀alkylene-aryl,-arylene-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo), —(C₃-C₈carbocyclo)-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo), and —(C₃-C₈heterocyclo)-C₁-C₁₀alkyl, where aryl on R₁₀ comprising aryl isoptionally substituted with [R₇]_(h);

h is 1, 2, 3, 4 or 5; and

X is O or S;

provided that when R^(3A) is hydrogen X is S.

Another aspect of the invention relates to compound of formula IIIa:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or            aralkyl; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂,—CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl;

R¹¹ is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z is

R⁷ is independently selected for each occurrence from the groupconsisting of F, Cl, I, Br, NO₂, CN and CF₃;

R¹⁰ is hydrogen, —C₁-C₁₀alkyl, —C₃-C₈-carbocycle, aryl,—C₁-C₁₀heteroalkyl, —C₃-C₈heterocyclo, —C₁-C₁₀alkylene-aryl,-arylene-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo), —(C₃-C₈carbocyclo)-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo), and —(C₃-C₈heterocyclo)-C₁-C₁₀alkyl, where aryl on R₁₀ comprising aryl isoptionally substituted with [R₇]_(h);

h is 1, 2, 3, 4 or 5; and

X is O or S.

R⁶ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl; and

h is 1, 2, 3, 4 or 5.

Another aspect of the invention relates to compound of formula In:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ i,

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or -(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z is

L is an antibody;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or            aralkyl; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

or R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

R⁶ is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl;

R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄ aryl;

R¹³ is C₁-C₁₀ heterocyclyl; and

X is O or S;

provided that when R^(3A) is hydrogen X is S.

Another aspect of the invention relates to compound of formula IIIb:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or            aralkyl; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂,—CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl;

R¹¹ is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z is

L is an antibody;

X is O or S.

Another aspect of the invention relates to compound of formula IIc:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

R^(1′) is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z′ is

L is an antibody;

D is —C(R^(4A′))(R^(4B′))— or is absent;

R^(2′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, or is absent if

is present;

R^(3A′) and R^(3B′) are either of the following:

-   -   (i) R^(3A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B′) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or            aralkyl, or R^(3B′) is C₂-C₄ alkylene and forms 5-7 member            ring as indicated by            ; or    -   (ii) R^(3A′) and R^(3B′) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A′) and R^(4B′) are either of the following:

-   -   (i) R^(4A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A′) and R^(4B′) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

or R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

R⁶ is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl;

R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄ aryl;

R¹³ is C₁-C₁₀ heterocyclyl; and

X is O or S;

provided that when R^(3A) is hydrogen X is S.

Another aspect of the invention relates to compound of formula IIIc:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or            aralkyl; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂,—CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl;

R^(11′) is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or -(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z′ is

L is an antibody;

X is O or S.

Another aspect of the invention relates to compound of formula IId:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

L is an antibody;

[linker] is a divalent linker;

D is —C(R^(4A′))(R^(4B′))— or is absent;

R^(2′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, or is absent if

is present;

R^(3A′) and R^(3B′) are either of the following:

-   -   (i) R^(3A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B′) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or            aralkyl, or R^(3B′) is C₂-C₄ alkylene and forms 5-7 member            ring as indicated by            ; or    -   (ii) R^(3A′) and R^(3B′) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A′) and R^(4B′) are either of the following:

-   -   (i) R^(4A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A′) and R^(4B′) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀heterocyclyl;

or R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

R⁶ is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl;

R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄ aryl;

R¹³ is C₁-C₁₀ heterocyclyl; and

X is O or S;

provided that when R^(3A) is hydrogen X is S.

Another aspect of the invention relates to compound of formula formulaIIId:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or            aralkyl; or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂,—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂,—CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl;

[linker] is a divalent linker;

L is an antibody;

X is O or S.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein the compoundis represented by

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein W is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein W is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein W is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein W is

In certain embodiments of the invention W is:

W is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ ishydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ ishydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ is C₁-C₈alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ ismethyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R² ishydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R² ishydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R² is C₁-C₈alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R² ismethyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ ishydrogen; and R² is methyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ ismethyl; and R² is methyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(3B) is C₁-C₈ alkyl,C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl,heteroaralkyl, halogen or aralkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(3B) is C₁-C₈ alkyl,C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl,heteroaralkyl, aralkyl or halogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ishalogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ishydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) isC₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ismethyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3B) isC₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3B) ismethyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3B) isisopropyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3B) isC₃-C₈ carbocyclyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3B) iscylohexyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) isC₁-C₈ alkyl; and R^(3B) is C₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ismethyl; and R^(3B) is methyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ishydrogen; and R^(3B) is C₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) ishydrogen; and R^(3B) is isopropyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) andR^(3B) taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) andR^(3B) taken together are C₂-C₈ alkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) andR^(3B) taken together are —CH₂CH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) andR^(3B) taken together are —CH₂CH₂CH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) andR^(3B) taken together are —CH₂CH₂CH₂CH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) andR^(3B) taken together are C₁-C₈ heteroalkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(3A) andR^(3B) taken together are —CH₂OCH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(4B) is C₁-C₈ alkyl,C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl,heteroaralkyl or aralkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) ishydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) isC₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) ismethyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4B) ishydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4B) isC₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4B) ismethyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) isC₁-C₈ alkyl; and R^(4B) is C₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) ismethyl; and R^(4B) is methyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) ishydrogen; and R^(4B) is hydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) ishydrogen; and R^(4B) is C₁-C₈ alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) andR^(4B) taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) andR^(4B) taken together are C₂-C₈ alkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) andR^(4B) taken together are —CH₂CH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) andR^(4B) taken together are —CH₂CH₂CH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) andR^(4B) taken together are —CH₂CH₂CH₂CH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) andR^(4B) taken together are C₁-C₈ heteroalkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R^(4A) andR^(4B) taken together are —CH₂OCH₂—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁵ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁶ ishydrogen.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁶ is C₁-C₈alkyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁶ ismethyl.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein X is O.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein X is S.

In certain embodiments, the present invention relates to any of theaforementioned compounds, or a pharmaceutically acceptable salt orsolvate thereof, and attendant definitions, wherein the compound isselected from the group consisting of:

-   N-Methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N²-[(1-Aminocyclopentyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N-Methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2-phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide;-   N-Methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N-Methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N²-[(1-Aminocyclopentyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N²-[(1-Aminocyclopropyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1-Amino-N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]cyclohexanecarboxamide;-   2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1-phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N²-[(3-Aminooxetan-3-yl)carbonyl]-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide;-   N,2-Dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-Dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-Dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N²-(3-Amino-2,2-dimethylpropanoyl)-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N²-(3-Amino-2,2-dimethylpropanoyl)-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide;-   2-Methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[1-(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[bicyclo[4.2.0]octa-1,3,5-trien-7-yl(carboxy)methyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   2-methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3    oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   Methyl    N-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-(3-amino-2,2-dimethylpropanoyl)-L-valyl](methyl)amino}-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L-phenylalaninate;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(methylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-2-amino-1-benzyl-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-oxo-2-(propylamino)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{R1S)-1-benzyl-2-(diethylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(tert-butylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   3-methyl-D-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   3-methyl-L-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   L-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   D-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   1,2-dimethyl-L-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   1,2-dimethyl-D-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   N˜2˜-[2,2-dimethyl-3-(methylamino)propanoyl]-N-{(1S,2R)-2-methoxy-4-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide;-   Methyl    N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[2,2-dimethyl-3-(methylamino)propanoyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate;-   Methyl    N-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2S)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalaninate;-   Methyl    N-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2R)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalaninate;-   N-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2S)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine;-   N-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2R)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine;-   Methyl    N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate;-   Methyl    N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate;-   (2S)—N-[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide;-   (2R)—N-[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide;-   2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-2-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-N-methyl-L-valinamide;-   N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-2-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-N-methyl-L-valinamide;-   (2S)—N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide;-   (2R)—N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide;-   N-2-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N-2-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-(1,2,3,4-tetrahydroquinolin-6-yl)propan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanine;-   N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanine;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2R,4S)-4-carboxy-1-phenylpentan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-(bicyclo[1.1.1]pent-1-ylamino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(1R)-2-methoxy-2-oxo-1-(1-phenylcyclopropyl)ethyl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(1S)-2-methoxy-2-oxo-1-(1-phenylcyclopropyl)ethyl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(1R)-1-[(7R)-bicyclo[4.2.0]octa-1,3,5-trien-7-yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(1S)-1-[(7S)-bicyclo[4.2.0]octa-1,3,5-trien-7-yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(1S)-1-[(7R)-bicyclo[4.2.0]octa-1,3,5-trien-7-yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;

N,N,2-trimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;

-   N,N,2-trimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(R)-carboxy(1-phenylcyclopropyl)methyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   difluoro    {2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-7-benzyl-15-{2-[(3,5-dimethyl-1H-pyrrol-2-yl-kappaN)methylidene]-2H-pyrrol-5-yl-kappaN}-4-methyl-5,8,13-trioxo-2-oxa-6,9,12-triazapentadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamidato}boron;-   2-methyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   methyl    N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(3-aminooxetan-3-yl)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate;-   2-methylalanyl-N-{(3R,4S,5S)-1-[(2S)-2-{(3R,4R,7S,12S)-7-benzyl-14-[3-chloro-4-(propan-2-yloxy)phenyl]-4-methyl-[2-[4-(8-methylimidazo[1,2-a]pyridin-2-yl)benzyl]-5,8,14-trioxo-2,9-dioxa-6,13-diazatetradecan-3-yl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide;-   2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-{[4-(5-fluoro-1,3-benzothiazol-2-yl)-2-methylphenyl]amino}-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S)-1-oxo-3-phenyl-1-(prop-2-en-1-yloxy)propan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-({(2S)-1-oxo-3-phenyl-1-[(1H-1,2,3-triazol-4-ylmethyl)amino]propan-2-yl}amino)propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S)-1-oxo-3-phenyl-1-(prop-2-yn-1-ylamino)propan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-imidazol-4-yl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1R)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S)-1-oxo-3-phenyl-1-(piperazin-1-yl)propan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide;-   1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide;    and-   2-methyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R¹ is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Y is C₂-C₂₀alkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Y is—(CH₂)_(p)—; and p is 1-10.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 1. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 2. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 3. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 4. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 5. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 6. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 7. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is ₈. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 9. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein p is 10.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Y is C₂-C₂₀heteroalkylene.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Y is—(CH₂CH₂O)_(q)CH₂CH₂—; and q is 1-10.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 1. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 2. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 3. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 4. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 5. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 6. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 7. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 8. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 9. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein q is 10.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁷ is F orCl; and h is 4 or 5.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁷ is F; andh is 3, 4 or 5.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein R⁷ is F; andh is 5.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is —NH₂.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein G is Cl. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein G is Br. Incertain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein G is I.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein the compoundis selected from the group consisting of the compounds of Table 18B.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein the compoundis selected from the group consisting of:

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is —NHL.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein Z is

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein L is H(C)—.

In certain embodiments, the present invention relates to any of theaforementioned compounds and attendant definitions, wherein L is anantibody selected from a murine antibody for the treatment of ovariancancer such as oregovomab (OVAREX®); a murine IgG_(2a) antibody for thetreatment of colorectal cancer such as edrecolomab (PANOREX®); ananti-EGFR IgG chimeric antibody for the treatment of epidermal growthfactor positive cancers, such as head and neck cancer, for instancecetuximab (ERBITUX®); a humanized antibody for the treatment of sarcoma,such as a Humanized Monoclonal Antibody to the Vitronectin Receptor(α_(v)β₃) like Vitaxin®; a humanized IgG₁ antibody for the treatment ofchronic lymphocytic leukemia (CLL) such as alemtuzumab (CAMPATH I/H®);SMART ID10 which is a humanized anti-HLA-DR antibody for the treatmentof non-Hodgkin's lymphoma; 131I Lym-1 (ONCOLYM®)which is a radiolabeledmurine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin'slymphoma; a humanized anti-CD2 mAb for the treatment of Hodgkin'sDisease or non-Hodgkin's lymphoma such as ALLOMUNE®; labetuzumab(CEACIDE®) which is a humanized anti-CEA antibody for the treatment ofcolorectal cancer; bevacizumab (AVASTIN®) which is a humanizedanti-VEGF-A mAb for the treatment of brain, colon, kidney, or lungcancer; Ibritumomab tiuxetan (ZEVALIN®) which is an anti-CD20 monoclonalantibody to the treatment of non-Hodgkin's lymphoma; ofatumumab(ARZERRA®) which is a human anti-CD20 monoclonal antibody for thetreatment of chronic lymphocytic leukemia; panitumumab (VECTIBIX®) whichis a human anti-EGFR monoclonal antibody for the treatment of coloncancer; rituximab (RITUXAN®) which is an anti-CD20 chimeric monoclonalantibody for the treatment of chronic lymphocytic leukemia andnon-Hodgkin's lymphoma; tositumomab (BEXXAR®) which is an anti-CD20monoclonal antibody for the treatment of non-Hodgkin's lymphoma;trastuzumab (HERCEPTIN®) which is an anti-HER2 receptor monoclonalantibody for the treatment of breast and stomach cancer; ipilimumab(YERVOY®) which is an anti-CTLA4 human monoclonal antibody for thetreatment of melanoma; gemtuzumab and inotuzumab ozogamicin.

In another specific embodiment, L includes antibodies selected fromanti-I-13 antibodies, including anti-I-13 antibodies used in thetreatment of cancer, for instance anti-IL-13Rα2 antibodies.

In yet another specific embodiment, L includes antibodies selected fromanti-Notch antibodies, including anti-Notch antibodies used in thetreatment of cancer.

In certain embodiments, the antibody L is bound to the linker via asulfur bond or via a sulfur-sulfur bond.

Another aspect of the invention relates to an antibody drug conjugatecomprising any of the aforementioned compounds.

Another aspect of the invention relates to an antibody drug conjugatecomprising an antibody and any one of the aforementioned compounds.

In certain embodiments, the present invention relates to any of theaforementioned antibody drug conjugates and attendant definitions,wherein the compound is covalently bound to the antibody.

In certain embodiments, the present invention relates to any of theaforementioned antibody drug conjugates and attendant definitions,wherein the compound in said antibody drug conjugate is selected fromthe group consisting of the compounds of Table 18B,

In certain embodiments, the present invention relates to any of theaforementioned antibody drug conjugates and attendant definitions,wherein the antibody drug conjugate comprises between 2, 3, 4, 5, 6, 7,8, 9 or 10 compounds of the invention.

In certain embodiments, the present invention relates to any of theaforementioned antibody drug conjugates and attendant definitions,wherein the antibody drug conjugate comprises 3 or 4 compounds of theinvention.

The Antibody Unit (Ab)

As noted above, the term “antibody” (or “Ab”) herein is used in thebroadest sense and specifically covers intact monoclonal antibodies,polyclonal antibodies, monospecific antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments that exhibit thedesired biological activity. In addition, while certain aspects of theinvention described herein refer to antibody drug conjugates, it isfurther envisioned that the antibody portion of the conjugate might bereplaced with anything that specifically binds or reactively associatesor complexes with a receptor, antigen or other receptive moietyassociated with a given target-cell population. For example, instead ofcontaining an antibody a conjugates of the invention could contain atargeting molecule that binds to, complexes with, or reacts with areceptor, antigen or other receptive moiety of a cell population soughtto be therapeutically or otherwise biologically modified. Example ofsuch molecules include smaller molecular weight proteins, polypeptide orpeptides, lectins, glycoproteins, non-peptides, vitamins,nutrient-transport molecules (such as, but not limited to, transferrin),or any other cell binding molecule or substances. In certain aspects,the antibody or other such targeting molecule acts to deliver a drug tothe particular target cell population with which the antibody or othertargeting molecule interacts.

Heteroatoms that may be present on an antibody unit include sulfur (inone embodiment, from a sulfhydryl group of an antibody), oxygen (in oneembodiment, from a carbonyl, carboxyl or hydroxyl group of an antibody)and nitrogen (in one embodiment, from a primary or secondary amino groupof an antibody). These hetero atoms can be present on the antibody inthe antibody's natural state, for example a naturally-occurringantibody, or can be introduced into the antibody via chemicalmodification.

In one embodiment, an antibody unit has a sulfhydryl group and theantibody unit bonds via the sulfhydryl group's sulfur atom.

In another embodiment, the antibody has lysine residues that can reactwith activated esters (such esters include, but are not limited to,N-hydroxysuccinimde, pentafluorophenyl, and p-nitrophenyl esters) andthus form an amide bond consisting of the nitrogen atom of the antibodyunit and a carbonyl.

In yet another aspect, the antibody unit has one or more lysine residuesthat can be chemically modified to introduce one or more sulfhydrylgroups. The reagents that can be used to modify lysines include, but arenot limited to, N-succinimidyl S-acetylthioacetate (SATA) and2-Iminothiolane hydrochloride (Traut's Reagent).

In another embodiment, the antibody unit can have one or morecarbohydrate groups that can be chemically modified to have one or moresulfhydryl groups.

In yet another embodiment, the antibody unit can have one or morecarbohydrate groups that can be oxidized to provide an aldehyde group(see, e.g., Laguzza, et al., 1989, J. Med. Chem. 32(3):548-55). Thecorresponding aldehyde can form a bond with a reactive site such as, forexample, hydrazine and hydroxylamine. Other protocols for themodification of proteins for the attachment or association of drugs aredescribed in Coligan et al., Current Protocols in Protein Science, vol.2, John Wiley & Sons (2002) (incorporated herein by reference).

When the conjugates comprise non-immunoreactive protein, polypeptide, orpeptide units instead of an antibody, useful non-immunoreactive protein,polypeptide, or peptide units include, but are not limited to,transferrin, epidermal growth factors (“EGF”), bombesin, gastrin,gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6,transforming growth factors (“TOP”), such as TGF-α and TGF-β, vacciniagrowth factor (“VGF”), insulin and insulin-like growth factors I and II,somatostatin, lectins and apoprotein from low density lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of immunized animals. Useful monoclonalantibodies are homogeneous populations of antibodies to a particularantigenic determinant (e.g., a cancer cell antigen, a viral antigen, amicrobial antigen, a protein, a peptide, a carbohydrate, a chemical,nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to anantigen-of-interest can be prepared by using any technique known in theart which provides for the production of antibody molecules bycontinuous cell lines in culture.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art(e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312;Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982,Meth. Enzymol. 92:3-16).

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art and are discussed infra.

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to target cells (e.g.,cancer cell antigens, viral antigens, or microbial antigens) or otherantibodies that bind to tumor cells or matrix. In this regard,“functionally active” means that the fragment, derivative or analog isable to elicit anti-anti-idiotype antibodies that recognize the sameantigen that the antibody from which the fragment, derivative or analogis derived recognized. Specifically, in an exemplary embodiment theantigenicity of the idiotype of the immunoglobulin molecule can beenhanced by deletion of framework and CDR sequences that are C-terminalto the CDR sequence that specifically recognizes the antigen. Todetermine which CDR sequences bind the antigen, synthetic peptidescontaining the CDR sequences can be used in binding assays with theantigen by any binding assay method known in the art (e.g., the BIA coreassay) (for location of the CDR sequences, see, e.g., Kabat et al.,1991, Sequences of Proteins of Immunological Interest, Fifth Edition,National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J.Immunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, Fab fragments, Fvs, single chainantibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or anyother molecule with the same specificity as the antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as for example,those having a variable region derived from a murine monoclonal andhuman immunoglobulin constant regions. (See, e.g., U.S. Pat. No.4,816,567; and U.S. Pat. No. 4,816,397, which are incorporated herein byreference in their entirety.) Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule. (See, e.g., U.S. Pat. No.5,585,089, which is incorporated herein by reference in its entirety.)Such chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in International Publication No. WO 87/02671; European PatentPublication No. 0 184 187; European Patent Publication No. 0 171 496;European Patent Publication No. 0 173 494; International Publication No.WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No.012 023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987,Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985,Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986,BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler etal., 1988, J. Immunol. 141:4053-4060; each of which is incorporatedherein by reference in its entirety.

Completely human antibodies are particularly desirable and can beproduced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, 1995, Int. Rev.Immunol. 13:65-93. For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which isincorporated herein by reference in its entirety. Other human antibodiescan be obtained commercially from, for example, Abgenix, Inc. (nowAmgen, Freemont, Calif.) and Medarex (Princeton, N.J.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (See, e.g., Jespers et al., 1994,Biotechnology 12:899-903). Human antibodies can also be produced usingvarious techniques known in the art, including phage display libraries(see, e.g., Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks etal., 1991, J. Mol. Biol. 222:581; Quan and Carter, 2002, The rise ofmonoclonal antibodies as therapeutics, In Anti-IgE and Allergic Disease,Jardieu and Fick, eds., Marcel Dekker, New York, N.Y., Chapter 20, pp.427-469).

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not from an antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation,derivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

Antibodies can have modifications (e.g., substitutions, deletions oradditions) in amino acid residues that interact with Fc receptors. Inparticular, antibodies can have modifications in amino acid residuesidentified as involved in the interaction between the anti-Fc domain andthe FcRn receptor (see, e.g., International Publication No. WO 97/34631,which is incorporated herein by reference in its entirety).

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the artsuch as, e.g., chemical synthesis or recombinant expression techniques.The nucleotide sequence encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, literature publications, or by routine cloning andsequencing.

In a specific embodiment, known antibodies for the treatment of cancercan be used. Antibodies immunospecific for a cancer cell antigen can beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., recombinant expression techniques.

The nucleotide sequence encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, the literature publications, or by routine cloning andsequencing. Examples of antibodies available for the treatment of cancerinclude, but are not limited to, OVAREX® which is a murine antibody forthe treatment of ovarian cancer; PANOREX® (Glaxo Wellcome, NC) which isa murine IgG_(2a) antibody for the treatment of colorectal cancer;Cetuximab ERBITUX® (Imclone Systems Inc., NY) which is an anti-EGFR IgGchimeric antibody for the treatment of epidermal growth factor positivecancers, such as head and neck cancer; Vitaxin® (MedImmune, Inc., MD)which is a humanized antibody for the treatment of sarcoma; CAMPATH I/H®(Leukosite, MA) which is a humanized IgG₁ antibody for the treatment ofchronic lymphocytic leukemia (CLL); SMART ID10 (Protein Design Labs,Inc., CA) which is a humanized anti-HLA-DR antibody for the treatment ofnon-Hodgkin's lymphoma; ONCOLYM® (Techniclone, Inc., CA) which is aradiolabeled murine anti-HLA-Dr10 antibody for the treatment ofnon-Hodgkin's lymphoma; ALLOMUNE® (BioTransplant, CA) which is ahumanized anti-CD2 mAb for the treatment of Hodgkin's Disease ornon-Hodgkin's lymphoma; CEACIDE® (Immunomedics, NJ) which is a humanizedanti-CEA antibody for the treatment of colorectal cancer; AVASTIN®(Genentech/Roche, CA) which is a humanized anti-VEGF-A mAb for thetreatment of brain, colon, kidney, or lung cancer; ZEVALIN® (SpectrumPharmaceuticals, NV) which is an anti-CD20 monoclonal antibody to thetreatment of non-Hodgkin's lymphoma; ARZERRA® (GSK, UK) which is a humananti-CD20 monoclonal antibody for the treatment of chronic lymphocyticleukemia; VECTIBIX® (Amgen, CA) which is a human anti-EGFR monoclonalantibody for the treatment of colon cancer; RITUXAN® (Genentech/BioGen,CA) which is an anti-CD20 chimeric monoclonal antibody for the treatmentof chronic lymphocytic leukemia and non-Hodgkin's lymphoma; BEXXAR®(GSK, UK) which is an anti-CD20 monoclonal antibody for the treatment ofnon-Hodgkin's lymphoma; HERCEPTIN® (Genentech, CA) which is an anti-HER2receptor monoclonal antibody for the treatment of breast and stomachcancer; YERVOY® (BMS, NJ) which is an anti-CTLA4 human monoclonalantibody for the treatment of melanoma; MYLOTARG® (Wyeth/Pfizer, NY)which is anti-CD33 humanized monoclonal antibody conjugated tocalicheamicin for the treatment of acute myelogenous leukemia; and,inotuzumab ozogamicin (Wyeth/Pfizer, NY) which is an anti-CD22 humanizedmonoclonal antibody conjugated to calicheamicin for the treatment ofacute lymphocytic leukemia and non-Hodgkin's lymphoma.

In another specific embodiment, anti-IL13 antibodies, includinganti-IL13 antibodies used in the treatment of cancer, can be used.

In another specific embodiment, anti-Notch antibodies, includinganti-Notch antibodies used in the treatment of cancer, can be used.

In attempts to discover effective cellular targets for cancer diagnosisand therapy, researchers have sought to identify transmembrane orotherwise tumor-associated polypeptides that are specifically expressedon the surface of one or more particular type(s) of cancer cell ascompared to on one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Synthesis of Compounds and Antibody Drug Conjugates Thereof

The compounds and conjugates of the invention can be made using thesynthetic procedures outlined below in the Exemplification.

As described in more detail below, the compounds and conjugates of theinvention can be prepared using a section of a linker unit having areactive site for binding to the compound.

Linker

A linker (sometimes referred to as “[linker]” herein) is a bifunctionalcompound which can be used to link a drug and an antibody to form anantibody drug conjugate (ADC). Such conjugates are useful, for example,in the formation of immunoconjugates directed against tumor associatedantigens. Such conjugates allow the selective delivery of cytotoxicdrugs to tumor cells.

In one embodiment, the linker has the formula: is

wherein

Y is C₂-C₂₀ alkylene or C₂-C₂₀ heteroalkylene; C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z is

R⁷ is independently selected for each occurrence from the groupconsisting of F, Cl, I, Br, NO₂, CN and CF₃;

R¹⁰ is hydrogen, —C₁-C₁₀alkyl, —C₃-C₈-carbocycle, aryl,—C₁-C₁₀heteroalkyl, —C₃-C₈heterocyclo, —C₁-C₁₀alkylene-aryl,-arylene-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo), —(C₃-C₈carbocyclo)-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo), and —(C₃-C₈heterocyclo)-C₁-C₁₀alkyl, where aryl on R₁₀ comprising aryl isoptionally substituted with [R₇]_(h); and

h is 1, 2, 3, 4 or 5.

In an ADC the linker serves to attach the payload to the antibody.

In one aspect, a second section of the linker unit is introduced whichhas a second reactive site e.g., an electrophilic group that is reactiveto a nucleophilic group present on an antibody unit (e.g., an antibody).Useful nucleophilic groups on an antibody include but are not limitedto, sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a linker unit and forms a covalent bond to a linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups. The electrophilic group provides a convenient sitefor antibody attachment.

In another embodiment, a linker unit has a reactive site which has anucleophilic group that is reactive to an electrophilic group present onan antibody. Useful electrophilic groups on an antibody include, but arenot limited to, aldehyde and ketone carbonyl groups. The heteroatom of anucleophilic group of a linker unit can react with an electrophilicgroup on an antibody and form a covalent bond to the antibody. Usefulnucleophilic groups on a linker unit include, but are not limited to,hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. The electrophilic group on an antibodyprovides a convenient site for attachment to a linker unit.

Amino functional groups are also useful reactive sites for a linker unitbecause they can react with carboxylic acid, or activated esters of acompound to form an amide linkage. Typically, the peptide-basedcompounds of the invention can be prepared by forming a peptide bondbetween two or more amino acids and/or peptide fragments. Such peptidebonds can be prepared, for example, according to the liquid phasesynthesis method (see, e.g., Schroder and Lubke, “The Peptides”, volume1, pp 76-136, 1965, Academic Press) that is well known in the field ofpeptide chemistry.

As described in more detail below, the conjugates can be prepared usinga section of the linker having a reactive site for binding to a compoundof the invention and introducing another section of the linker unithaving a reactive site for an antibody. In one aspect, a linker unit hasa reactive site which has an electrophilic group that is reactive with anucleophilic group present on an antibody unit, such as an antibody. Theelectrophilic group provides a convenient site for antibody attachment.Useful nucleophilic groups on an antibody include but are not limitedto, sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a Linker unit and forms a covalent bond to a linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups.

In another embodiment, a linker unit has a reactive site which has anucleophilic group that is reactive with an electrophilic group presenton an antibody unit. The electrophilic group on an antibody provides aconvenient site for attachment to a linker unit. Useful electrophilicgroups on an antibody include, but are not limited to, aldehyde andketone carbonyl groups. The heteroatom of a nucleophilic group of alinker unit can react with an electrophilic group on an antibody andform a covalent bond to the antibody. Useful nucleophilic groups on alinker unit include, but are not limited to, hydrazide, oxime, amino,hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

As used herein, “mc-” previously known as “MalC-” refers to

As used herein, “mcValCitPABC-” previously known as “MalCValCitPABC-”refers to

As used herein, “Ma1PegXC2-” refers to

As used herein, “AmPegXC2-” refers to

As used herein, “mcValCitPABCAmPegXC2-” refers to

As used herein, “Ma1PegXC2ValCitPABC-” refers to

As used herein, “2BrAcPegXC2” refers to

As used herein, “mv-” refers to

As used herein, “mb-” refers to

As used herein, “me-” refers to

As used herein, “MalC6-” refers to

As used herein, “PFPCOPe2XC2ValCitPABC-” refers

As used herein, “PFPCOPegXC2AmPegYC2-” refers to

As used herein, “PFPCOPegXC2AlaAlaAsnPABC-” refers to

As used herein, “PFPCOPegXC2-” refers to

As used herein, “PFPCOPegXC2AmPegYC2PABC-” refers to

As used herein, “mcGly-” refers to

As used herein, “AzCOC2Ph4AmCOPeg2C2-” refers to

As used herein, “AzCOC2Ph4AmPeglC1-” refers to

As used herein, “AcLysValCitPABC-” refers to

Conjugation with Transglutaminase

In certain embodiments, a compound of the invention may be covalentlycrosslinked to an Fc-containing or Fab-containing polypeptide engineeredwith an acyl donor glutamine-containing tag (e.g., Gln-containingpeptide tags or Q-tags) or an endogenous glutamine made reactive (i.e.,the ability to form a covalent bond as an acyl donor in the presence ofan amine and a transglutaminase) by polypeptide engineering (e.g., viaamino acid deletion, insertion, substitution, mutation, or anycombination thereof on the polypeptide), in the presence oftransglutaminase, provided that the compound of the invention comprisesan amine donor agent (e.g., small molecule comprising or attached to areactive amine), thereby forming a stable and homogenous population ofan engineered Fc-containing polypeptide conjugate with the amine donoragent being site-specifically conjugated to the Fc-containing orFab-containing polypeptide through the acyl donor glutamine-containingtag or the exposed/accessible/reactive endogenous glutamine. Forexample, compounds of the invention may be conjugated as described inInternational Patent Application Serial No. PCT/IB2011/054899, whoseentire contents are incorporated herein by reference. In certainembodiments, to facilitate conjugation of the compound of the inventionto an Fc-containing or Fab-containing polypeptide engineered with anacyl donor glutamine-containing tag or an endogenous glutamine madereactive by polypeptide engineering in the presence of transglutaminase,Z is NH₂.

Conjugation to the Human Light Chain Kappa Domain Constant Region

In certain embodiments, a compound of the invention may be covalentlyattached to the side chain of K¹⁸⁸ of the human light chain kappa domainconstant region (CLκ) (full light chain numbering according to Kabat).K188 may also be termed CLκ K80, when counting only the human kappaconstant region, for example, of SEQ ID NOs: 1, 2, 3 and 4).

For example, compounds of the invention may be conjugated as describedin U.S. patent application Ser. No. 13/180,204, or WO2012/007896 whoseentire contents are incorporated herein by reference. In certainembodiments, to facilitate conjugation to K188 CLκ (CLκ-K80), Z is

R⁷ is independently selected for each occurrence from the groupconsisting of F, Cl, I, Br, NO₂, CN and CF₃; and h is 1, 2, 3, 4 or 5.

Tn certain embodiments, to facilitate conjugation to K¹⁸⁸ CLκ (CLκ-K80),Z is

The present invention further provides antibody drug conjugatescomprising an antibody, or antigen binding portions thereof, comprisinga constant kappa domain covalently conjugated to a toxin of theinvention, characterized in that at least one toxin of the invention iscovalently conjugated to K80 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 orSEQ ID NO: 4 (Table 1). In some aspects, the number of toxins of theinvention covalently conjugated to the at K80 may be a range whose lowerlimit is selected from the group consisting of about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, and about 2.0, and whose upper limit isselected from the group consisting of about 2.0, about 2.1, about 2.2,about 2.3, about 2.4, about 2.5. In some aspects, p is about 2.

Conjugation of the toxin with the constant light domain of an antibodyis particularly desirable to minimize, or prevent, any interference withbinding of the Fc portion of the antibody to Fc receptors (such as FcγRand FcRn) or binding of the antibody to its respective target.Conversely, conjugation of the respective toxin to the Fc portion of anantibody may decrease the antibody half-life in vivo and/or its capacityto interact with the immune system (effector function). Conjugation ofthe toxin in the variable heavy chain (VH) or variable light chain (VL)region of the antibody carries a risk of diminishing the binding of theantibody to its cognate.

Furthermore, whereas conjugation to CLκ-K80 is reliable and robust,conjugation to other antibody surface lysines, each of slightlydifferent reactivity and pI can result in an heterogeneous sample ofconjugated antibodies that can release conjugated molecules atinopportune or irregular times, such as during circulation and prior todelivery of the Effector Moiety to the target by antibody recognition.

In addition, the present invention provides for known polymorphisms ofthe kappa chain V/A at position 45 and A/L at position 83 (giving the 3identified human constant kappa polymorphisms Km(1):V45/L83 (SEQ IDNO:2), Km(1,2): A45/L83 (SEQ ID NO:3), and Km(3) A45/V83 (SEQ ID NO:4)).The variability of residues at positions 45 and 83 in SEQ ID NO:1 may beselected so as to only provide for any one, two or all three of theKm(1), Km(1,2), and Km(3) polymorphisms.

TABLE 1  SEQ ID NO DESCRIPTION SEQUENCE 1 hLC constantTVAAPSVFIF PPSDEQLKSG TASVVCLLNN region GENUSFYPREAKVQW KVDNxLQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKxYACEVTHQGLSSPVTKS FNRGEC 2 h LC constant TVAAPSVFIF PPSDEQLKSG TASVVCLLNNregion Km(1) FYPREAKVQW KVDNVLQSGN SQESVTEQDS polymorphismKDSTYSLSST LTLSKADYEK HKLYACEVTH (V45/L83) QGLSSPVTKS FNRGEC 3h LC constant TVAAPSVFIF PPSDEQLKSG TASVVCLLNN region Km(1, 2)FYPREAKVQW KVDNALQSGN SQESVTEQDS polymorphismKDSTYSLSST LTLSKADYEK HKLYACEVTH A45/L83 QGLSSPVTKS FNRGEC 4h LC constant TVAAPSVFIF PPSDEQLKSG TASVVCLLNN region Km(3)FYPREAKVQW KVDNALQSGN SQESVTEQDS polymorphismKDSTYSLSST LTLSKADYEK HKVYACEVTH A45/V83 QGLSSPVTKS FNRGECWherein x at position 45 is A or V, and x at position 83 is L or V.

In certain embodiments, the invention provides for a compositioncomprising a compound of the invention covalently conjugated to anantibody (or antigen binding portion thereof), wherein at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% of the compound of the invention in thecomposition is conjugated to the antibody or antigen binding portionthereof at K¹⁸⁸ CLκ.

In certain embodiments, the compounds of the invention

may be conjugated to the combining site of a catalytic antibody, such asaldolase antibodies, or antigen binding portion thereof. Aldolaseantibodies contain combining site portions that, when unencumbered (forexample by conjugation), catalyze an aldol addition reaction between analiphatic ketone donor and an aldehyde acceptor. The contents of USPatent Application Publication No. US 2006/205670 are incorporatedherein by reference, in particular pages 78-118 describing linkers, andparagraphs [0153]-[0233] describing antibodies, useful fragments,variants and modifications thereof, h38C2, combining sites andcomplimentary determining regions (CDRs), and related antibodytechnology (Table 2, and exemplary compounds below):

The term “combining site” includes the CDRs and the adjacent frameworkresidues that are involved in antigen binding.

TABLE 2  SEQ DESCRIP- ID NO TION SEQUENCE 5 h38C2 VLELQMTQSPSS LSASVGDRVT ITCRSSQSLL HTYGSPYLNW YLQKPGQSPK LLIYKVSNRFSGVPSRFSGS GSGTDFTLTI SSLQPEDFAV YFCSQGTHLP YTFGGGTKVE IK 6 h38C2 VHEVQLVESGGG LVQPGGSLRL SCAASGFTFS NYWMSWVRQS PEKGLEWVSE IRLRSDNYATHYAESVKGRF TISRDNSKNT LYLQMNSLRA EDTGIYYCKT YFYSFSYWGQ GTLVTVSS 7h38C2 LC ELQMTQSPSS LSASVGDRVT ITCRSSQSLLHTYGSPYLNW YLQKPGQSPK LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAVYFCSQGTHLP YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAKVQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPVTKSFNRGEC 8 h38C2 HC EVQLVESGGG LVQPGGSLRL SCAASGFTFSNYWMSWVRQS PEKGLEWVSE IRLRSDNYAT HYAESVKGRF TISRDNSKNT LYLQMNSLRAEDTGIYYCKT YFYSFSYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDYFPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNTKVDKRVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHEDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALPAPIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKConjugation with Linkers Comprising Succinimides, Including Ring-OpenedVersions

In certain embodiments, the present invention includes a compound of theinvention conjugated via a succinimide-based linker or a ring-openedsuccinimide-based linker. The stability of the succinimide-cysteinelinkage has become an area of increasing interest. Succinimides can betransferred both in vitro and in vivo to exogenous thiol nucleophiles,presumably through a retro-Michael reaction resulting in a maleimidethat is subsequently attacked by a thiol. It is believed that hydrolysisof the ring results in a species that is resistant to the retro-Michaelreaction. This renders the resulting conjugate more stable andpotentially more efficacious. Conditions may be optimized to forciblyopen the succinimide ring on the conjugate. Basic conditions resulted infacile hydrolysis of the ring. For instance, linkers containing apolyethylene glycol (PEG) chain can be hydrolysed at pH 9.2 at 37° C. inapproximately 12 h, and linkers containing an alkyl chain, such as “mc”may require a higher temperature and longer reaction time in order todrive the ring-opening to completion.

Example of Forced Hydrolysis of Maleimide-Based Conjugates

In order to assess the stability of these conjugates and prioritizesamples for in vivo evaluation, an assay was developed that involves thetreatment of the maleimide-linked conjugates with excess aqueousglutathione (GSH) or plasma. Aliquots of the reaction mixture wereanalyzed at various timepoints to determine the loading of theconjugate, using the methodology described above. The results (Table 24)indicate that the drug-antibody linkage is slowly cleaved in aGSH-dependent manner. As expected, the rate of cleavage is highlydependent upon the hydrolysis of the succinimide ring. Importantly,these results appear to translate to improved PK exposure, as measuredby an increase in area-under-curve (AUC) of the conjugate and by anincrease in the conjugate/Ab exposure ratio.

Method for Assessing the Stability of ADCs

The ADC sample (30 ng) in PBS is mixed with glutathione (GSH) solutionto produce final concentration of GSH of 0.5 mM and 3 mg/mL proteinconcentration. A control sample (without GSH) was likewise prepared from30 ng ADC diluted to 3 mg/mL in PBS. The GSH-treated ADC sample and thecontrol ADC sample were incubated at 37° C. and were sampled at 0, 3,and 6 days. Aliquots were reduced with excess TCEP, acidified by adding0.1% formic acid solution with 10% acetonitrile and analyzed by forloading by LC/MS as described below.

Sample analysis: Analysis was performed using an Aglient 1100 capillaryHPLC coupled with Waters Xevo G2 Q-TOF mass spectrometer. The analyteswere loaded onto a Zorbax Poroshell 300SB C8 column (0.5 mm×75 mm,maintained at 80° C.) with 0.1% formic acid, and eluted using a gradientof 20-40% buffer B (80% acetonitrile, 18% 1-propanol, 2% water with 0.1%formic acid) at a flow rate of 20 μl/min over 5.5 minutes. Massspectrometric detection was carried out in positive, sensitivity modewith capillary voltage set at 3.3 kV. Data analysis was performed withMaxEnt 1 function in MassLynx and intensities were used for loadingcalculation based on the previously described formula.

Compositions and Methods of Administration

In other embodiments, another aspect of the invention relates topharmaceutical compositions including an effective amount of a compoundof the invention and/or antibody drug conjugate thereof and apharmaceutically acceptable carrier or vehicle. In certain embodiments,the compositions are suitable for veterinary or human administration.

The present pharmaceutical compositions can be in any form that allowsfor the composition to be administered to a patient. For example, thecomposition can be in the form of a solid or liquid. Typical routes ofadministration include, without limitation, parenteral, ocular andintra-tumor. Parenteral administration includes subcutaneous injections,intravenous, intramuscular or intrasternal injection or infusiontechniques. In one aspect, the compositions are administeredparenterally. In a specific embodiment, the compositions areadministered intravenously.

Pharmaceutical compositions can be formulated so as to allow a compoundof the invention and/or antibody drug conjugate thereof to bebioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where forexample, a tablet can be a single dosage unit, and a container of acompound of the invention and/or antibody drug conjugate thereof inliquid form can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the a compound of the invention and/orantibody drug conjugate thereof , the manner of administration, and thecomposition employed.

The pharmaceutically acceptable carrier or vehicle can be solid orparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) can be liquid. In addition, the carriers)can be particulate.

The composition can be in the form of a liquid, e.g., a solution,emulsion or suspension. In a composition for administration byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates,phosphates or amino acids and agents for the adjustment of tonicity suchas sodium chloride or dextrose. A parenteral composition can be enclosedin ampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of a compound of the invention and/or antibody drug conjugatethereof that is effective in the treatment of a particular disorder orcondition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques. In addition, in vitroor in vivo assays can optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the compositions willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances.

The compositions comprise an effective amount of a compound of theinvention and/or antibody drug conjugate thereof such that a suitabledosage will be obtained. Typically, this amount is at least about 0.01%of a compound of the invention and/or antibody drug conjugate thereof byweight of the composition. In an exemplary embodiment, pharmaceuticalcompositions are prepared so that a parenteral dosage unit contains fromabout 0.01% to about 2% by weight of the amount of a compound of theinvention and/or antibody drug conjugate thereof.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of a compound of the invention and/or antibody drugconjugate thereof per kg of the patient's body weight. In one aspect,the composition can include from about 1 to about 100 mg of a compoundof the invention and/or antibody drug conjugate thereof per kg of thepatient's body weight. In another aspect, the amount administered willbe in the range from about 0.1 to about 25 mg/kg of body weight of acompound of the invention and/or antibody drug conjugate thereof.

Generally, the dosage of a compound of the invention and/or antibodydrug conjugate thereof administered to a patient is typically about 0.01mg/kg to about 20 mg/kg of the patient's body weight. In one aspect, thedosage administered to a patient is between about 0.01 mg/kg to about 10mg/kg of the patient's body weight. In another aspect, the dosageadministered to a patient is between about 0.1 mg/kg and about 10 mg/kgof the patient's body weight. In yet another aspect, the dosageadministered to a patient is between about 0.1 mg/kg and about 5 mg/kgof the patient's body weight. In yet another aspect the dosageadministered is between about 0.1 mg/kg to about 3 mg/kg of thepatient's body weight. In yet another aspect, the dosage administered isbetween about 1 mg/kg to about 3 mg/kg of the patient's body weight.

A compound of the invention and/or antibody drug conjugate thereof canbe administered by any convenient route, for example by infusion orbolus injection. Administration can be systemic or local. Variousdelivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used toadminister a compound of the invention and/or antibody drug conjugatethereof. In certain embodiments, more than one compound of the inventionand/or antibody drug conjugate thereof is administered to a patient.

In specific embodiments, it can be desirable to administer one or morecompounds of the invention and/or antibody drug conjugates thereoflocally to the area in need of treatment. This can be achieved, forexample, and not by way of limitation, by local infusion during surgery;topical application, e.g., in conjunction with a wound dressing aftersurgery; by injection; by means of a catheter; or by means of animplant, the implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as silastic membranes, or fibers. Inone embodiment, administration can be by direct injection at the site(or former site) of a cancer, tumor or neoplastic or pre-neoplastictissue. In another embodiment, administration can be by direct injectionat the site (or former site) of a manifestation of an autoimmunedisease.

In yet another embodiment, the compound of the invention and/or antibodydrug conjugate thereof can be delivered in a controlled release system,such as but not limited to, a pump or various polymeric materials can beused. In yet another embodiment, a controlled-release system can beplaced in proximity of the target of the compound of the inventionand/or antibody drug conjugate thereof, e.g., the liver, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer(Science 249:1527-1533 (1990)) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a compound or antibody drug conjugate thereof is administered.Such pharmaceutical carriers can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin. Thecarriers can be saline, and the like. In addition, auxiliary,stabilizing and other agents can be used. In one embodiment, whenadministered to a patient, the compound or conjugate andpharmaceutically acceptable carriers are sterile. Water is an exemplarycarrier when the compound or conjugate are administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions. Thepresent compositions, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, pellets,powders, sustained-release formulations, or any other form suitable foruse. Other examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

In an embodiment, the compound of the invention and/or antibody drugconjugate thereof are formulated in accordance with routine proceduresas a pharmaceutical composition adapted for intravenous administrationto animals, particularly human beings. Typically, the carriers orvehicles for intravenous administration are sterile isotonic aqueousbuffer solutions. Where necessary, the compositions can also include asolubilizing agent. Compositions for intravenous administration canoptionally comprise a local anesthetic such as lignocaine to ease painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where a compound of the invention and/or antibody drugconjugate thereof is to be administered by infusion, it can bedispensed, for example, with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the compound of theinvention and/or antibody drug conjugate thereof is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

Whether in solid or liquid form, the present compositions can include apharmacological agent used in the treatment of cancer.

Therapeutics Uses of Compounds and Antibody Drug Conjugates Thereof

Another aspect of the invention relates to a method of using thecompounds of the invention and antibody drug conjugates thereof fortreating cancer.

The compounds of the invention and/or antibody drug conjugates thereofare useful for inhibiting the multiplication of a tumor cell or cancercell, causing apoptosis in a tumor or cancer cell, or for treatingcancer in a patient. The compounds of the invention and/or antibody drugconjugates thereof can be used accordingly in a variety of settings forthe treatment of animal cancers. Said conjugates can be used to delivera compound of the invention to a tumor cell or cancer cell. Withoutbeing bound by theory, in one embodiment, the antibody of the conjugatebinds to or associates with a cancer-cell or a tumor-cell-associatedantigen, and the conjugate can be taken up (internalized) inside a tumorcell or cancer cell through receptor-mediated endocytosis or otherinternalization mechanism. The antigen can be attached to a tumor cellor cancer cell or can be an extracellular matrix protein associated withthe tumor cell or cancer cell. In certain embodiments, once inside thecell, one or more specific peptide sequences are enzymatically orhydrolytically cleaved by one or more tumor cell or cancercell-associated proteases, resulting in release of a compound of theinvention from the conjugate. The released compound of the invention isthen free to migrate within the cell and induce cytotoxic or cytostaticactivities. The conjugate also can be cleaved by an intracellularprotease to release a compound of the invention. In an alternativeembodiment, the compound of the invention is cleaved from conjugateoutside the tumor cell or cancer cell, and the compound of the inventionsubsequently penetrates the cell.

In certain embodiments, the conjugates provide conjugation-specifictumor or cancer drug targeting, thus reducing general toxicity of thecompounds of the invention.

In another embodiment, the antibody unit binds to the tumor cell orcancer cell.

In another embodiment, the antibody unit binds to a tumor cell or cancercell antigen which is on the surface of the tumor cell or cancer cell.

In another embodiment, the antibody unit binds to a tumor cell or cancercell antigen which is an extracellular matrix protein associated withthe tumor cell or cancer cell.

The specificity of the antibody unit for a particular tumor cell orcancer cell can be important for determining those tumors or cancersthat are most effectively treated.

Particular types of cancers that can be treated with a compound of theinvention and/or antibody drug conjugate thereof, include but are notlimited to, carcinomas of the bladder, breast, cervix, colon,endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, skin,stomach, and testes; and blood born cancers including but not limited toleukemias and lymphomas.

Multi-Modality Therapy for Cancer.

Cancers, including, but not limited to, a tumor, metastasis, or otherdisease or disorder characterized by uncontrolled cell growth, can betreated or inhibited by administration of a compound of the inventionand/or antibody drug conjugate thereof.

In other embodiments, methods for treating cancer are provided,including administering to a patient in need thereof an effective amountof a compound of the invention and/or antibody drug conjugate thereofand a chemotherapeutic agent. In one embodiment the chemotherapeuticagent is that with which treatment of the cancer has not been found tobe refractory. In another embodiment, the chemotherapeutic agent is thatwith which the treatment of cancer has been found to be refractory. Acompound of the invention and/or antibody drug conjugate thereof can beadministered to a patient that has also undergone surgery as treatmentfor the cancer.

In some embodiments, the patient also receives an additional treatment,such as radiation therapy. In a specific embodiment, the compound of theinvention and/or antibody drug conjugate thereof is administeredconcurrently with the chemotherapeutic agent or with radiation therapy.In another specific embodiment, the chemotherapeutic agent or radiationtherapy is administered prior or subsequent to administration of acompound of the invention and/or antibody drug conjugate thereof.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents, such a standardof care chemotherapeutic agent(s), can be administered.

Additionally, methods of treatment of cancer with a compound of theinvention and/or antibody drug conjugate thereof are provided as analternative to chemotherapy or radiation therapy where the chemotherapyor the radiation therapy has proven or can prove too toxic, e.g.,results in unacceptable or unbearable side effects, for the subjectbeing treated. The patient being treated can, optionally, be treatedwith another cancer treatment such as surgery, radiation therapy orchemotherapy, depending on which treatment is found to be acceptable orbearable.

The compounds of the invention and/or antibody drug conjugates thereofcan also be used in an in vitro or ex vivo fashion, such as for thetreatment of certain cancers, including, but not limited to leukemiasand lymphomas, such treatment involving autologous stem celltransplants. This can involve a multi-step process in which the animal'sautologous hematopoietic stein cells are harvested and purged of allcancer cells, the animal's remaining bone-marrow cell population is theneradicated via the administration of a high dose of a compound of theinvention and/or antibody drug conjugate thereof with or withoutaccompanying high dose radiation therapy, and the stem cell graft isinfused back into the animal. Supportive care is then provided whilebone marrow function is restored and the patient recovers.

Released Species

Further embodiments of the invention include the chemical speciesreleased, inside or in the vicinity of the cancer cell or tumor cell bywhat is believed to be enzymatic and/or hydrolytic cleavage by one ormore cancer cell or tumor cell-associated proteases. Such compoundsinclude the species described herein, and also include compounds such asthose described in the structure:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is

R¹ is

Y is C₂-C₂₀ alkylene or C₂-C₂₀ heteroalkylene; C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or -(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-;

Z″ is

Z′″ is

G is halogen, —OH, —SH or —S—C₁-C₆ alkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl;

R^(3A) and R^(3B) are defined as either of the following:

-   -   (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl        or halogen; and        -   R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,            C₁-C₁ heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl;            or    -   (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R^(4A) and R^(4B) are defined as either of the following:

-   -   (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈        carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or        aralkyl; and        -   R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈            carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or            aralkyl; or    -   (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or        C₁-C₈ heteroalkylene;

R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

or R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl;

R⁶ is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl;

R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄ aryl;

R¹³ is C₁-C₁₀ heterocyclyl; and

R⁷ is independently selected for each occurrence from the groupconsisting of F, Cl, I, Br, NO₂, CN and CF₃;

R¹⁰ is hydrogen, —C₁-C₁₀alkyl, —C₃-C₈-carbocycle, aryl,—C₁-C₁₀heteroalkyl, —C₃-C₈heterocyclo, —C₁-C₁₀alkylene-aryl,-arylene-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo), —(C₃-C₈carbocyclo)-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo), and —(C₃-C₈heterocyclo)-C₁-C₁₀alkyl, where aryl on R₁₀ comprising aryl isoptionally substituted with [R₇]_(h);

h is 1, 2, 3, 4 or 5; and

X is O or S.

Of particular interest are compounds of formula IV having thestructures:

The invention is further described in the following examples, which arenot intended to limit the scope of the invention.

EXEMPLIFICATION

Experiments were generally carried out under inert atmosphere (nitrogenor argon), particularly in cases where oxygen- or moisture-sensitivereagents or intermediates were employed. Commercial solvents andreagents were generally used without further purification, includinganhydrous solvents where appropriate (generally Sure-Seal™ products fromthe Aldrich Chemical Company, Milwaukee, Wis.). Products were generallydried under vacuum before being carried on to further reactions orsubmitted for biological testing. Mass spectrometry data is reportedfrom either liquid chromatography-mass spectrometry (LCMS), atmosphericpressure chemical ionization (APCI) or gas chromatography-massspectrometry (GCMS) instrumentation. Chemical shifts for nuclearmagnetic resonance (NMR) data are expressed in parts per million (ppm,δ) referenced to residual peaks from the deuterated solvents employed.

For syntheses referencing procedures in other Examples or Methods,reaction Protocol (length of reaction and temperature) may vary. Ingeneral, reactions were followed by thin layer chromatography (TLC) ormass spectrometry, and subjected to work-up when appropriate.Purifications may vary between experiments: in general, solvents and thesolvent ratios used for eluants/gradients were chosen to provideappropriate Rfs or retention times.

Optical rotations were performed on a Perkin-Elmer polarimeter 343(Serial number 9506).

HRMS were performed on an Agilent 6220 TOF LC/MS.

Compound names were generated with ACD Labs software.

HPLC and LC-MS Conditions Used for Analysis

Protocol A:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.1%formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 100% B over8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL/minute.Temperature: 25° C.; Detection: DAD 215 nm, 254 nm; MS (+) range150-2000 daltons; Injection volume: 10 μL; Instrument: Agilent 1200LCMS.

Protocol B:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.1%formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 50% B over 1.5 minutes, 50% to 100% B over6.5 minutes, then 100% B over 3 minutes; Flow rate: 0.75 mL/minute.Temperature: 25° C.; Detection: DAD 215 nm; MS (+) range 150-2000daltons; Injection volume: 10 μL; Instrument: Agilent 1200 LCMS.

Protocol C:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in methanol (v/v); Gradient: 50% to 100% B over 10minutes; Flow rate: 0.75 mL/minute. Temperature: not controlled;Detection: DAD 215 nm, 254 nm; Injection volume: 10 μL; Instrument:Agilent 1100 HPLC.

Protocol D:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in methanol (v/v); Gradient: 5% to 100% B over 8minutes; Flow rate: 0.75 mL/minute. Temperature: not controlled;Detection: DAD 215 nm, 254 nm; Injection volume: 10 μL; Instrument:Agilent 1100 HPLC.

Protocol E:

Column: Phenomenex Lux Amylose-2, 250×4.6 mm, 5 μm; Mobile phase A:heptane; Mobile phase B: ethanol (denaturated); Gradient: 5% to 100% Bover 10 minutes; Flow rate: 1.5 mL/minute. Temperature: not controlled;Detection: DAD 215 nm, 254 nm; MS (+) range 150-1500 daltons; Injectionvolume: 10 μL; Instrument: Agilent 1100 LCMS.

Protocol F:

Column: Waters Acquity UPLC BEH, C18, 2.1×50 mm, 1.7 μm; Mobile phase A:0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 0.7minute, 95% B over 0.1 minute; Flow rate: 1.25 mL/minute. Temperature:60° C.; Detection: 200-450 nm; MS (+) range 100-1200 daltons; Injectionvolume: 5 μL; Instrument: Waters Acquity.

Protocol G:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 100% B over8.5 minutes; Flow rate: 1.5 mL/minute. Temperature: not controlled;Detection: DAD 210 nm; Injection volume: 10 μL; Instrument: Agilent 1100HPLC.

Protocol H:

Column: Phenomenex Gemini-NX, C18, 4.6×50 mm, 3 μm, 110 Å; Mobile phaseA: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 0% to 100% B over 4.10 minutes, linearthen 100% B over 0.4 minute; Flow rate: 1.5 mL/minute. Temperature: 60°C.; Detection: DAD 200-450 nm; MS (+) range 100-2000 daltons; Injectionvolume: 5 μL; Instrument: Agilent.

Protocol I:

Column: Atlantis T3, 75×3.0 mm, 3 μm; Mobile phase A: 0.05%trifluoroacetic acid in water (v/v); Mobile phase B: acetonitrile;Gradient: 5% to 95% B over 5.75 minutes; Flow rate: 1.2 mL/minute.Temperature: 45° C.; Detection: DAD 215 nm, 230 nm, 254 nm; MS (+)range: 150-1200 daltons; Injection volume: 5 μL; Instrument: Agilent1100 LCMS.

Protocol J:

Column: Phenomenex Luna Phenyl-Hexyl, 150×3.0 mm, 5 μm; Mobile phase A:0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 100% B over8.5 minutes, then 100% B over 1 minute; Flow rate: 0.75 mL/minute.Temperature: 25° C.; Detection: DAD 215 nm, 254 nm; MS (+) range150-2000 daltons; Injection volume: 10 μL; Instrument: Agilent 1200LCMS.

Protocol K:

Column: Symmetry-C18, 50×2.1 mm, 3.5 μm; Mobile phase A: 0.1% formicacid in water (v/v); Mobile phase B: 0.1% formic acid in methanol (v/v);Gradient: 10% to 90% B over 6.5 minutes; Flow rate: 0.7 mL/minute.Temperature: room temperature; Detection: DAD 215 nm; MS (+) range100-1500 daltons; Injection volume: 3 μL; Instrument: Waters 996 PDA.

Protocol L:

Column: XBridge C-18, 150×4.6 mm, 3.5 μm; Mobile phase A: 5 mM aqueousammonium acetate solution; Mobile phase B: acetonitrile; Gradient: 10% Bover 3 minutes then 10% to 80% B over 14 minutes; Flow rate: 0.7mL/minute. Temperature: room temperature; Detection: DAD 215 nm; MS (+)range 100-1500 daltons; Injection volume: 3 μL; Instrument: Waters 996PDA.

Protocol M:

Column: Phenomenex Luna, 150×3.0 mm, 5 μm; Mobile phase A: 0.1% formicacid in water (v/v); Mobile phase B: 0.1% formic acid in methanol (v/v);Gradient: 50% B over 1.5 minutes, 50% to 80% B over 8.5 minutes, then80% B over 10 minutes; Flow rate: 0.75 mL/minute. Temperature: 45° C.;Detection: DAD 215 nm, 254 nm; MS (+) range 90-2000 daltons; Injectionvolume: 10 μL; Instrument: Agilent 1200 LCMS.

Protocol N:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 100% B over23.5 minutes; Flow rate: 1.5 mL/minute. Temperature: not controlled;Detection: DAD 210 nm; Injection Volume: 10 μL; Instrument: Agilent 1100HPLC

Protocol O:

Column: Column: Agilent Poroshell 300SB-C8, 75×2.1 mm, 2.6 μm; Mobilephase A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formicacid in acetonitrile (v/v); Gradient: 20% B to 45% B over 4 minutes;Flow rate: 1.0 mL/minute. Temperature: 60° C.; Detection: 220 nm; MS (+)range 400-2000Da; Injection volume: 10 μL; Instrument: Agilent 1100 LC,Waters MicromassZQ MS. Deconvolution was performed using MaxEntl.

Protocol P:

Column: Column: TSK-gel G3000SWx1, 300×7.8 mm, 10 μm; Mobile phase:Phosphate buffer saline (PBS, 1×), pH 7.4 with 2% acetonitrile;Isocratic; Flow rate: 1 mL/minute. Temperature: room temperature;Injection Volume: 5 μL; Instrument: Agilent 1100 HPLC.

Protocol Q:

Column: Waters Acquity UPLC HSS T3, C18, 2.1×50 mm, 1.7 μm; Mobile phaseA: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 2.5minutes, 95% B over 0.35 minute; Flow rate: 1.25 mL/minute. Temperature:60° C.; Detection: 200-450 nm; MS (+) range 100-2000 daltons; Injectionvolume: 5 μL; Instrument: Waters Acquity.

Protocol Q1:

Column: Waters Acquity UPLC HSS T3, C18, 2.1×50 mm, 1.7 μm; Mobile phaseA: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 1.5minute, 95% B over 0.35 minute; Flow rate: 1.25 mL/minute. Temperature:60° C.; Detection: 200-450 nm; MS (+) range 100-2000 daltons; Injectionvolume: 5 μL; Instrument: Waters Acquity.

Protocol Q2:

Column: Xtimate C18, 2.1×30 mm, 3 μm; Mobile phase A: 0.1%trifluoroacetic acid in water (v/v); Mobile phase B: 0.1%trifluoroacetic acid in acetonitrile (v/v); Gradient: 10% to 80% B over0.9 minutes, 80% B over 0.6 minutes; 100% B for 0.5 minutes; Flow rate:1.2 mL/minute. Detection: DAD 220 nM; Temperature: 25° C.; Injectionvolume: 1 μL; Instrument: Agilent.

Protocol Q3:

Column: Xtimate C18, 2.1×30 mm, 3 μm; Mobile phase A: 0.1%trifluoroacetic acid in water (v/v); Mobile phase B: 0.1%trifluoroacetic acid in acetonitrile (v/v); Gradient 0% to 60% B over0.9 minutes, 60% B over 0.6 minutes; 100% B for 0.5 minutes; Flow rate:1.2 mL/minute. Detection: DAD 220 nM; Temperature: 25° C.; Injectionvolume: 1 μl; Instrument: Agilent.

Protocol R:

Column: Phenomenex Luna, 150×3.0 mm, 5 nm; Mobile phase A: 0.1% formicacid in water (v/v); Mobile phase B: 0.1% formic acid in methanol (v/v);Gradient: 5% B over 1.5 minutes, 5% to 100% B over 8.5 minutes, then100% B over 1 minute; Flow rate: 0.75 mL/minute. Temperature: 45° C.;Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 daltons; Injectionvolume: 10 mL; Instrument: 305 RP Agilent 1200 LCMS.

Protocol S:

Column: Phenomenex Luna, 150×3.0 mm, 5 nm; Mobile phase A: 0.1%trifluoroacetic acid in water (v/v); Mobile phase B: 0.1%trifluoroacetic in acetonitrile (v/v); Gradient: 5% B over 1.5 minutes,5% to 95% B over 8.5 minutes, then 100% B over 1 minute; Flow rate: 1.0mL/minute. Temperature: not controlled; Detection: DAD 210 nm; MS (+)range 150-2000 daltons; Injection volume: 10 mL; Instrument: 305 RPAgilent 1100 HPLC.

Protocol T:

Column: Atlantis dC18, 50×4.6 mm, 5 nm; Mobile phase A: 0.05%trifluoroacetic acid in water (v/v); Mobile phase B: 0.05%trifluoroacetic acid in acetonitrile (v/v); Gradient: 5% to 95% B over4.0 minutes; then hold at 95% B over 1 minute; Flow rate: 2 mL/minute.Temperature: room temperature; Detection: DAD 215 nm; MS (+) range160-1000 daltons; Injection volume: 3 mL; Instrument: Waters 996 PDA.

Protocol U:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 nm; Mobile phase A: 0.1%formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 100% B over8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL/minute.Temperature: 45° C.; Detection: DAD 215 nm, 254 nm; MS (+) range150-2000 daltons; Injection volume: 10 mL; Instrument: Agilent 1200LCMS.

Protocol V:

Column: HPLC-V Ultimate XB-C18, 50×3.0 mm, 3 nm; Mobile phase A: 0.225%trifluoroacetic acid in water (v/v); Mobile phase B: 0.225%trifluoroacetic acid in acetonitrile (v/v); Gradient: 30% to 90% B over6 minutes; Flow rate: 1.2 mL/minute. Temperature: 40° C.; Detection: DAD220 nm; Injection volume: 1 mL; Instrument: SHIMADZU.

Protocol W:

Column: HPLC-V Ultimate XB-C18, 50×3.0 mm, 3 nm; Mobile phase A: 0.1%trifluoroacetic acid in water (v/v); Mobile phase B: 0.1%trifluoroacetic acid in acetonitrile (v/v); Gradient: 10% to 80% B over6 minutes; Flow rate: 1.2 mL/minute. Temperature: 40° C.; Detection: DAD220 nm; Injection volume: 3 μL; Instrument: SHIMADZU.

Protocol X:

Column: YMC-pack ODS-A, 150×4.6 mm, 5 μm; Mobile phase A: 0.1%trifluoroacetic acid in water (v/v); Mobile phase B: 0.1%trifluoroacetic acid in acetonitrile (v/v); Gradient: 10% to 80% B over6 minutes; Flow rate: 1.2 mL/minute. Detection: DAD 220 nm. Temperature:40° C.; Injection volume: 3 μL; Instrument: SHIMADZU.

Protocol Y:

Column: YMC-pack ODS-A, 150×4.6 mm, 5 μm; Mobile Phase A: 0.1%trifluoroacetic acid in water (v/v); Mobile Phase B: 0.1%trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 95% B over10 minutes, then 95% B for 5 minutes; Flow rate^(.) 1.5 mL/minute;Detection: DAD 220 nm; Instrument: Agilent 1100.

Protocol Z:

Column: Xtimate C18, 2.1×30 μm, 3 μm; Mobile Phase A: 0.1%trifluoroacetic acid in water (v/v); Mobile phase B: 0.1%trifluoroacetic acid in acetonitrile (v/v); Gradient: 0% to 60% B over 2minutes; Flow rate: 1.2 ml/min. Temperature: 50° C.; Detection: 220 nm,MS (+) range 100-1000 daltons; Injection volume: 1 μL; Instrument:SHIMADZU.

Protocol AB:

Column: Phenomenex Luna C18 (2), 150×2.0 mm, 5 μm; Mobile phase A: 0.1%formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% to 100% B over 10 minutes, then 100% Bfor 2 minute; Flow rate: 0.5 mL/minute. Temperature: 25° C.; Detection:DAD 210 nm, 254 nm; MS (+) range 150-2000 daltons; Injection volume: 5μL; Instrument: Agilent 1100 LCMS.

Protocol BB:

Column: Phenomenex Luna C18 (2), 150×2.0 mm, 5 μm; Mobile phase A: 0.1%formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 2.0 minutes, 5% to 100% B over12 minutes, and 100% B for 2 minute, then 100% to 5% B over 1.5 min;Flow rate: 0.75 mL/minute. Temperature: 25° C.; Detection: DAD 215 nm,254 nm; MS (+) range 150-2000 daltons; Injection volume: 5 μL;Instrument: Agilent.

Protocol CB:

Column: Waters XBridge C18, 4.6×50 mm, 5 μm; Mobile phase A: 0.03%ammonium hydroxide in water (v/v); Mobile phase B: 0.03% ammoniumhydroxide in acetonitrile (v/v); Gradient: 5% to 95% B over 4.0 minutes,then 95% B for 1 minute; Flow rate: 2 mL/minute. Temperature: 25° C.;Detection: DAD 215 nm, MS (+) range 160-1000 daltons; Injection volume:4 μL; Instrument: Waters ZQ/Alliance 2795 HPLC.

Protocol DB:

Column: Waters Atlantis dC18, 4.6×50 mm, 5 μm; Mobile phase A: 0.05%trifluoroacetic acid in water (v/v); Mobile phase B: 0.05%trifluoroacetic acid in acetonitrile (v/v); Gradient: 5.0% to /95% Bover 4.0 minutes, then 95% B for 1 minute. Flow rate: 2 mL/minute.Temperature: 25° C.; Detection: DAD 215 nm, MS (+) range 160-1000daltons; Injection volume: 4 μL; Instrument: Waters ZQ/Alliance 2795HPLC.

Protocol EB:

Column: XBridge RP18, 2.1×50 mm, 5 μm; Mobile phase A: 0.02% ammoniumhydroxide in water (v/v); Mobile phase B: 0.02% ammonium hydroxide inacetonitrile (v/v); Gradient 10% to 80% B % over 6 minutes, then 80% for2 minutes; Flow rate: 1.2 mL/minute. Detection: DAD 220 nm.;Temperature: 50° C.

Protocol FB:

Column: Phenomenex Luna C18 (2), 150×2.0 mm, 5 μm; Mobile phase A: 0.1%formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 2.0 minutes, 5% to 100% B over10 minutes, and 100% B for 2 minute; Flow rate: 0.50 mL/minute.Temperature: 25° C.; Detection: DAD 215 nm, 254 nm; MS (+) range150-2000 daltons; Injection volume: 5 μL; Instrument: Agilent 1200 LCMS.

In some instances some minor alterations to analysis LC-MS and HPLCconditions were made such as but not limited a change in gradient orflow rate which is indicated by the symbol *.

HPLC Conditions Used for Purification

Method A:

Column: Phenomenex Lux Amylose-2, 250×21.2 mm, 5 μm; Mobile phase A:Heptane; Mobile phase B: Ethanol (denatured); Gradient: 5% to 100% Bover 6 min; Flow Rate: 27 mL/minute; Detection: DAD 210-360 nm; MS (+)range 150-2000 daltons; Instrument: Waters FractionLynx.

Method B:

Column: Phenomenex Luna C18(2), 150×21.2 mm, 5 μm; Mobile phase A: 0.02%acetic acid in water; Mobile phase B: 0.02% acetic acid in acetonitrile;Gradient: 5% B over 1.5 minutes, 5% to 45% B over 8.5 minutes; Flowrate: 27 mL/minute; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000daltons; Instrument: Waters FractionLynx.

Method C:

Column: Phenomenex Luna C18, 100×30 mm, 10 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in methanol (v/v); Gradient: 10% to 90% B over 20minutes; Flow rate: 20 mL/minute. Temperature: not controlled;Detection: DAD 210 nm, 254 nm; Injection Volume: variable; Instrument:Gilson.

Method D:

Column: Phenomenex Synergi Max-RP, 150×21.2 mm, 4 μMI; Mobile phase A:0.1% formic acid in water; Mobile phase B: 0.1% formic acid inacetonitrile; Gradient: 30% B for 1.5 minutes, 30% to 60% B over 8.5minutes, 60 to 100% B over 0.5 minutes then 100% B over 2 minutes; Flowrate: 27 mL/minute; Detection: DAD 210-360 nm; MS (+) range 150-2000daltons; Instrument: Waters FractionLynx.

Method E1:

Column: Phenomenex Luna C18(2), 150×21.2 mm, 5 μm; Mobile phase A: 0.1%formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile;Gradient: 40% B for 1.5 minutes, 40% to 80% B over 8.5 minutes, 80 to100% B over 0.5 minute then 100% B over 2 minutes; Flow rate: 27mL/minute; Detection: Detection: DAD 210-360 nm; MS (+) range 150-2000daltons; Instrument: Waters FractionLynx LCMS.

Method E2:

Column: Phenomenex Luna Phenyl-hexyl, 150×21.2 mm, 5 μm. The rest of theProtocols are identical to those described for Method E1.

Method F:

Column: Phenomenex Synergi Max-RP, 150×21.2 mm, 4 μm; Mobile phase A:0.1% formic acid in water; Mobile phase B: 0.1% formic acid in methanol;Gradient: 44% B for 1.5 minutes, 44% to 77% B over 8.5 minutes, then 77%B over 10 minutes; Flow rate: 27 mL/minute; Detection: DAD 210-360 nm;MS (+) range 150-2000 daltons; Instrument: Waters FractionLynx LCMS.

Method G:

Column: PrincetonSFC 2-ethylpyridine, 250×21.2 mm, 5 μm; Mobile phase A:heptane; Mobile phase B: ethanol (denaturated); Gradient: 1% B for 1.5minutes, 1% to 50% B over 8.5 minutes; Flow rate: 27 mL/minute;Detection: DAD 210-360 nm; MS (+) range 150-2000 daltons; Instrument:Waters FractionLynx LCMS.

Method H:

Column: Phenomenex Luna C18(2), 150×21.2 mm, 5 μm; Mobile phase A: 0.02%acetic acid in water; Mobile phase B: 0.02% acetic acid in acetonitrile;Gradient: 20% B over 1.5 minutes, 20% to 60% B over 10.5 minutes; Flowrate: 27 mL/minute; Detection: DAD 210-360 nm; MS (+) range 150-2000daltons; Instrument: Waters FractionLynx LCMS.

Method I:

Column: Phenomenex Luna C18(2), 150×21.2 mm, 5 μm; Mobile phase A: 0.1%formic acid in water; Mobile phase B: 0.1% formic acid in methanol;Gradient: 40% B over 1.5 minutes, 40% to 70% B over 8.5 minutes then 70%B over 10 minutes; Flow rate: 27 mL/minute; Detection: DAD 210-360 nm;MS (+) range 150-2000 daltons; Instrument: Waters FractionLynx LCMS.

Method J:

Column: Phenomenex Luna C18, 100×30 mm, 5 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in acetonitrile (v/v); Gradient: 10% to 90% B over20 minutes; Flow rate: 20 mL/minute. Temperature: not controlled;Detection: DAD 210 nm, 254 nm; Injection Volume: variable; Instrument:Gilson.

Method K:

Column: Phenomenex Luna C18(2), 150×21.2 mm, 5 μm; Mobile phase A: 0.1%formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile;Gradient: 20% B for 1.5 minutes, 20% to 50% B over 8.5 minutes, 50 to100% B over 0.5 minute then 100% B over 2 minutes; Flow rate: 27mL/minute; Detection: Detection: DAD 210-360 nm; MS (+) range 150-2000daltons; Instrument: Waters Fraction Lynx LCMS.

Method L:

Column: Phenomenex Luna C18(2), 150×21.2 mm, 5 μm; Mobile phase A: 0.1%formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile;Gradient: 30% B for 1.5 minutes, 30% to 50% B over 8.5 minutes, 50 to100% B over 0.5 minute then 100% B over 2 minutes; Flow rate: 27mL/minute; Detection: Detection: DAD 210-360 nm; MS (+) range 150-2000daltons; Instrument: Waters Fraction Lynx LCMS.

Method M:

Column: Waters Sunfire, C18, 19×100 mm, 5 μm; Mobile phase A: 0.05%trifluoroacetic acid in water (v/v); Mobile phase B: 0.05%trifluoroacetic acid in acetonitrile (v/v); Gradient: 0 to 100% over 8.5minutes. Flow rate 25 mL/minute. Detection: DAD 215 nm MS (+) range160-1000 daltons; Instrument: Waters FractionLynx.

Method N:

Column: Waters Sunfire, C18, 19×100 mm, 5 μm; Mobile phase A: 0.05%formic acid in water (v/v); Mobile phase B: 0.05% formic acid inacetonitrile (v/v); Gradient: 0 to 100% over 8.5 minutes. Flow rate 25mL/minute. Detection: DAD 215 nm MS (+) range 160-1000 daltons;Instrument: Waters FractionLynx.

Method O:

Column: Phenomenex Luna C18, 21.2×150 mm, 5 μm; Mobile phase A: 0.1%formic acid in water (v/v) acid in water (v/v); Mobile phase B: 0.1%formic acid in acetonitrile (v/v); Gradient(v/v); Gradient 20% B over1.5 minutes, 20% to 40% B over 8.5 minutes, 40 to 100% B over 0.5minutes, then hold 100% B for 1.5 minutes. Flow rate: 27 mL/minute.Detection: DAD 210-360 nm; MS (+) range 150-2000 daltons; Instrument:Waters FractionLynx.

Method P:

Column: Phenomenex Gemini C18, 21.2×250 mm, 5 μm; Mobile phase A: 0.225%ammonia hydroxide in water (pH 10) (v/v); Mobile phase B: 0.225% ammoniahydroxide in acetonitrile (v/v); Gradient: 45% to 85% B over 10 minutes.Flow rate 35 mL/minute. Detection: DAD 220 nm MS (+) range 100-1200daltons; Instrument Shimadzu MS Trigger.

Method Q:

Column: Column. Phenomenex Synergi C18, 50×250 mm, 10 μm; Mobile phaseA: 0.1% trifluoroacetic acid in water (v/v) Mobile phase B: Acetonitrile; Gradient 10% to 40% B over 25 minutes. Flow rate 100 mL/minute.Detection: UV/Vis 220 nm; Instrument: Shimadzu LC-8A.

Method R:

Column: Phenomenex Luna C18 (2), 250×21.2 mm, 5 μm; Mobile phase A: 0.1%TFA in water (v/v); Mobile phase B: 0.1% TFA in acetonitrile (v/v);Gradient: 10% to 100% over 30 minutes; Flow rate variable. Temperature:25° C.; Detection: DAD 215 nm, 254 nm; MS (+) range 150-2000 daltons;Injection volume: 1.8 mL: Instrument: Agilent 1100 Prep HPLC.

In some instances some minor alterations to purification conditions weremade such as but not limited to a change in gradient or flow rate whichis indicated by the symbol *.

General Procedures

General Procedure A:

Fmoc removal using diethylamine or piperidine. To a solution of theFmoc-containing compound in dichloromethane or N,N-dimethylformamide(also referred to as DMF), was added an equal volume of diethylamine orpiperidine. Reaction progress was monitored by LC-MS (or HPLC or TLC).Solvents were removed in vacuo, and in some cases the residue wasazeotroped one to four times with heptane. Residue was usually dilutedwith dichloromethane and a small amount of methanol before being reduceddown onto silica and purified by chromatography on silica gel, elutingwith methanol in dichloromethane (or other appropriate mixture ofsolvents) to afford the desired material (or crude material was used asis).

General Procedure B:

Boc removal or t-Bu ester cleavage using trifluoroacetic acid. To asolution of the Boc-containing compound or tert-butyl ester-containingcompound in dichloromethane at 0° C. (or at room temperature) was addedtrifluoroacetic acid, to afford a ratio of 1:4 trifluoroaceticacid:dichloromethane. Reaction progress was monitored by LC-MS (or HPLCor TLC). Solvents were removed in vacuo. The residue was azeotropedthree times with heptane to afford the desired material.

General Procedure C:

Boc removal or tert-butyl ester (also refers to t-Bu ester) cleavageusing hydrochloric acid in dioxane. To either a solution ofBoc-containing compound or tert-butyl ester-containing compound indioxane (or in some cases no solution, or other relevant solvent) wasadded a 4 M solution of hydrochloric acid in dioxane. Reaction progresswas monitored by LC-MS (or HPLC or TLC). The reaction was concentratedin vacuo and in some cases azeotroped one to four time with heptanes.

General Procedure D:

Coupling with O-(7-azabenzotriazol-1-yl)-N,N,N;N′-tetramethyluroniumhexafluorophosphate (HATU). To a stirring solution of the amine (1.0eq.) and acid (1.0-2.0 eq.) in dichloromethane, N,N-dimethylformamide(also referred to as DMF), or a mixture of both, HATU (1.0-2.0 eq.) wasadded followed by triethylamine (2.0-4.0 eq.) or diisopropylethylamine(2.0-4.0 eq., also referred to as Hunig's base). Reaction progress wasmonitored by LC-MS (or HPLC or TLC); the reaction was usually completedwithin three hours. Solvents were removed in vacuo. The residue waspurified by silica gel or reverse phase chromatography or in some casesazeotroped three times with heptanes, diluted with a small amount ofethyl acetate before being reduced down onto silica or C18 bonded silicaand purified by silica gel or reverse phase chromatography.

General Procedure E:

coupling withN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(MalcValCitPABC-PNP). To a mixture of the payload amine (1 eq.) andN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(MalcValCitPABC-PNP, Eur. Pat. Appl. (1994), EP624377, 1.0-2.0 eq.) inN,N-dimethylformamide or dimethylacetamide (also referred to as DMA),pyridine (0.0-4.0 eq.), diisopropylethylamine (0.0-4.0 eq.),2,6-dimethylpyridine (0.0-4.0 eq., also referred to as 2,6-Luditine) and1-hydroxybenzotriazole hydrate (0.01-1.1 eq. also referred to as HOBT)or 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol (0.01-1.1 eq., also referred toas HOAT) was added. After stirring at 40° C.-50° C. for 1-48 hours, thereaction mixture was concentrated in vacuo and azeotroped three timeswith heptane. The crude material was purified by reverse phasechromatography according to the specified method to afford the desiredmaterial.

General Procedure F:

Conjugation of commercial HERCEPTIN® antibody with linker-payload viainternal disulfides. Commercially available HERCEPTIN® antibody(Genentech Inc) was dialyzed into Dulbecco's Phosphate Buffered Saline(DPBS, Lonza). The dialyzed antibody was reduced with addition of xequivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 mMin distilled water) and diluted to 15 mg/mL final antibody concentrationusing DPBS, 5 mM 2,2′,2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid(EDTA), pH 7.0-7.4 (Buffer A). The reaction was incubated at 37° C. for1-2 hours and then cooled to room temperature. Conjugation was performedby addition of y equivalents of linker-payload (5-10 mM indimethylacetamide (DMA)). DMA was added to achieve 10-20% (v/v) totalorganic solvent component in final reaction mixture, and Buffer A addedto achieve 10 mg/mL final antibody concentration. The reaction wasincubated for 1-2 hours at room temperature. The reaction mixture wasthen buffer exchanged into DPBS (pH 7.4) using GE Healthcare SephadexG-25 M buffer exchange columns per manufacturer's instructions. Crudematerial was purified by size exclusion chromatography (SEC) using GEAKTA Explorer system with GE Superdex column and PBS (pH 7.4) eluent.

General Procedure G:

Conjugation reactions were performed in the upper portion of acentrifugal ultrafiltration device such as Amicon Ultra 50k Ultracelfilters (part #UFC805096, GE). A 132 mM stock solution of L-cysteine wasprepared in PBS containing 50 mM EDTA. This solution (50 μL) was addedto a mixture of the respective mutant antibody (5 mg) in 950 μL of PBScontaining 50 mM EDTA. The final cysteine concentration in the reactionmixture was 6.6 mM. After allowing the reaction to stand at roomtemperature (about 23° C.) for 1.5 hours the reaction tube wascentrifuged to concentrate the material to approximately 100 μL. Themixture was diluted to 1 mL with PBS containing 50 mM EDTA. This processwas repeated 4 times in order to remove all the cysteine reductant. Theresulting material was diluted to 1 mL in PBS containing 50 mM EDTA andtreated with 16 μL of a 5 mM solution of the maleimide linker-payload(from Table 18A in dimethyl acetamide (DMA) (approximately 5equivalents). After standing at room temperature (about 23° C.) for 1.5hours the reaction tube was centrifuged to concentrate the material toapproximately 100 μL. The mixture was diluted to 1 mL with PBS. Thisprocess was repeated 2 times in order to remove the excess maleimidereactant. The antibody conjugates were generally purified by sizeexclusion chromatography (SEC) using GE AKTA Explorer system with a GESuperdex200 column and PBS (pH7.4) eluent. The loading of the drug ontothe intended site of conjugation was determined using a variety ofmethods including mass spectrometry (MS), reverse phase HPLC, andhydrophobic interaction chromatography (HIC), as has been describedelsewhere. The reported value (in Tables 19A and 19B) is generallyobtained by LC-MS under reducing conditions.

General Procedure H:

A 20 mM TCEP solution (generally 50 to 100 molar equivalents) was addedto the antibody (typically 5 mg) such that the final antibodyconcentration was 5 mg/mL in PBS containing 50 mM EDTA. After allowingthe reaction to stand at 37° C. for 1.5 hours, the antibody was bufferexchanged into PBS containing 50 mM EDTA using a 50 kD MW cutoff spinconcentration device (3×3 mL wash, 10× concentration per cycle).Alternative methods such as TFF or dialysis are also useful inparticular circumstances. The resulting antibody was re-suspended in 1mL of PBS containing 50 mM EDTA and treated with a freshly prepared 50mM solution of DHA (dehydroascorbate) in 1:1 PBS/EtOH (final DHAconcentration is typically 1 mM) and allowed to stand at 4° C.overnight. The antibody/DHA mixture was buffer exchanged into PBScontaining 50 mM EDTA using a 50 kD MW cutoff spin concentration device(3×3 mL wash, 10× concentration per cycle). The resulting antibody wasre-suspended in 1 mL of PBS containing 50 mM EDTA and treated with 10 mMmaleimide linker-payload in DMA (typically 5-10 equivalents). Afterstanding for 1.5 hours, the material was buffer exchanged (as above)into 1 mL of PBS (3×3 mL washes, 10× concentration per cycle).Purification by SEC (as described previously) was performed as needed toremove any aggregated material.

General Procedure I:

The initial conjugation of the linker-payload was performed using thepreviously described method (General Procedure F). The resultingantibody-drug-conjugate was buffer exchanged into a 50 mM borate buffer(pH 9.2) using an ultrafiltration device (50 kd MW cutoff). Theresulting solution was heated to either 37° C. for 24 hours (for themaleimide-Peg linkers) or to 45° C. for 48 hours (for themaleimide-caproyl linkers). The resulting solution was cooled,buffer-exchanged into PBS, and purified by SEC (as described previously)in order to remove any aggregated material. LCMS analysis of thematerial indicated that the succinimide ring had completely opened (90%or more). Note that in examples where a methyl ester is present in thepayload, the ester is hydrolyzed to the carboxylic acid under thedescribed conditions.

General Procedure J:

The pentafluorophenyl esters were conjugated to the shown antibodyfollowing the procedure previously outlined in WO2012007896 A1

General Procedure K:

The conjugation of amino-alkyl linkers was accomplished viaenzyme-mediated ligation as described in WO2012059882 A2.

General Procedure L:

N-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-1-(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(prepared in the same manner as #136) was coupled to the relevant aminoacid or amine moiety using HATU (1.0-2.0 eq.) in the presence of Hunig'sbase (1.-5.0 eq.) in a solution of DMF, dichloromethane, or in somecases a solution of both (or a solution of one or more solvents).Reaction was monitored by LC-MS (or TLC or HPLC). Reaction wasconcentrated in vacuo and purified usually by silica chromatography orby prep HPLC. Fmoc protection was then removed as described in generalprocedure A followed by concentration in vacuo and purified by silicachromatography or by prep HPLC.

General Procedure M.

#151 was coupled to the relevant amine using HATU (1.0-2.0 eq., or otherappropriate coupling reagent) in the presence of Hunig's base (1.0-5.0eq.) in a solution of DMF, dichloromethane, or in some cases a solutionof both (or a solution of one or more solvents). Reaction was monitoredby LC-MS (or TLC or HPLC). Reaction was concentrated in vacuo. Bocde-protection was then performed as described in general procedure B,concentrated in vacuo and purified by silica chromatography or by prepHPLC.

General Procedure N.

1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-oicacid (or other appropriate Fmoc-AmPegXC2—COOH) was coupled to therelevant cytotoxic pentapeptide (or the cytotoxic pentapeptidecontaining a protecting group on a reactive moiety other than theN-terminus) using HATU (1.0-2.0 eq., or other appropriate couplingreagent) in the presence of Hunig's base (1.0-5.0 eq. or otherappropriate base) in a solution of DMF, dichloromethane, or in somecases a solution of both (or a solution of one or more solvents).Reaction was monitored by LC-MS (or TLC or HPLC). Reaction wasconcentrated in vacuo. Fmoc de-protection was performed according togeneral procedure A. In some cases a second de-protection was performedin order to remove a protecting group on a reactive moiety on thecytotoxic pentapeptide using general procedure B (or other relevantprocedure known in the literature based on the protecting group). Thereaction was concentrated in vacuo and purified by silica chromatographyor by prep HPLC.

General Procedure O.

The appropriate Fmoc-AmPegXC2—COOH is coupled to the relevant cytotoxicpentapeptide (or the cytotoxic pentapeptide containing a protectinggroup on a reactive moiety other than the N-terminus) and Fmocde-protection is performed according to general procedure N. Thereaction is concentrated in vacuo and then purified by silicachromatography or prep HPLC (or the crude material can be used as is).The appropriate PABC sequence (such as mcValCitPABC, or derivative of)is then installed according to general procedure E. In some cases if aprotecting group is present on cytotoxic pentapeptide portion of themolecule de-protection is then performed using general procedure A orgeneral procedure B (or other relevant procedure known in the literaturebased on the protecting group). The reaction is concentrated in vacuoand purified by silica chromatography or by prep HPLC.

General Procedure P.

Followed procedure E replacing mcValCitPABC-PNP, withMalPeg3C2ValCitPABC-PNP (prepared in a similar manner tomcValCitPABC-PNP).

General Procedure Q.

The appropriate Fmoc-AmPegXC2-COOH is coupled to the relevant cytotoxicpentapeptide (or the cytotoxic pentapeptide containing a protectinggroup on a reactive moiety other than the N-terminus) and Fmocde-protection is performed as described in general procedure N. Thereaction is concentrated in vacuo and then purified by silicachromatography or by prep HPLC (or the crude material can be used asis). To a stirring solution of this residue in DMF at 0° C. (or aslightly higher temperature in some cases) bromoacetic acid (1.0-2.0eq.) was added followed by Hunig's base (1.0-5.0 eq.) and HATU (1.0-2.0eq.) The reaction was allowed to warm to room temperature and stir atroom temperature while being monitored by LC-MS (or TLC or HPLC).Reaction was concentrated in vacuo and purified by prep HPLC.

General Procedure R.

Followed procedure E replacing mcValCitPABC-PNP, withN-(6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexanoyl)-L-valyl-N-5˜-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(prepared in a similar manner to mcValCitPABC-PNP). Fmoc de-protectionwas then performed (general procedure B) followed by prep HPLCpurification.

General Procedure S.

To a stirring solution of6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanal (1.0-3.0 eq.) inmethanol the relevant cytotoxic pentapeptide (1.0 eq) was added followedby formic acid. The reaction was allowed to stir at room temperature for1-40 minutes followed by the addition of sodium(cyano-kappaC)(trihydrido)borate(1-) (3.0-6.0 eq., also referred to assodium cyanborohydride). The reaction was monitored by LC-MS (or TLC orHPLC). In some cases additional6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanal (1.0-3.0 eq.) was added.The reaction was concentrated in vacuo followed by purification usingprep HPLC.

General Procedure T.

4-[3-oxo-3-(2-oxoazetidin-1-yl)propyl]anilinium is prepared as describedin the literature (Bioorganic and Medicinal Chemistry Letters. 2012,vol. 22, #13, 4249-4253) which is then coupled to bis(pentafluorophenyl)3,3′-[ethane-1,2-diylbis(oxy)]dipropanoate using HATU in dichloromethanefollowed by coupling to the desired cytotoxic pentapeptide. Material isthen purified by prep HPLC.

General Procedure U.

Fmoc-ValCitPABC-PNP is coupled to the desired cytotoxic pentapeptidefollowing general procedure E and then Fmoc is removed following generalprocedure A. This residue is then coupled to[2-oxo-2-({4-[3-oxo-3-(2-oxoazetidin-1-yl)propyl]phenyl}amino)ethoxy]acetic acid (which is prepared by coupling4-[3-oxo-3-(2-oxoazetidin-1-yl)propyl]anilinium with1,4-dioxane-2,6-dione following general procedure D). Material is thenpurified by prep HPLC.

General Procedure V.

Bis(pentafluorophenyl) 3,3′-[ethane-1,2-diylbis(oxy)]dipropanoate orbis(pentafluorophenyl) 4,7,10,13,16-pentaoxanonadecane-1,19-dioate iscoupled to the desired cytotoxic pentapeptide (or in some cases coupledto the desired cytotoxic pentapeptide containing a protecting group on areactive moiety other than the N-terminus) following general procedureD. If a protecting group is present, the protecting group is thenremoved (using relevant procedures described in the literature).Material is then purified by prep HPLC.

General Procedure W.

4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azamidecan-13-oate is coupledto the desired cytotoxic pentapeptide following general procedure E.Fmoc is removed following general procedure A. Bis(pentafluorophenyl)3,3′-[ethane-1,2-diylbis(oxy)]dipropanoate is coupled to this residuefollowing general procedure D. Material is then purified by prep HPLC.

General Procedure X1.

N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanyl-L-alanyl-N˜1˜-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-N˜4˜-trityl-L-aspartamideis coupled to the desired cytotoxic pentapeptide following generalprocedure E. Fmoc is removed following general procedure A and tritylprotecting group is removed following general procedure B.Bis(pentafluorophenyl) 3,3′-[ethane-1,2-diylbis(oxy)]dipropanoate iscoupled to this residue following general procedure D. Material ispurified by prep HPLC.

General Procedure X2.

N-{3-[2-(3-ethoxy-3-oxopropoxy)ethoxy]propanoyl}-L-valyl-N˜5˜-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamideis coupled to the desired cytotoxic pentapeptide following generalprocedure E. Ethyl ester is removed using lithium hydroxide in THF andwater. NHS ester is then formed by coupling residue with1-hydroxypyrrolidine-2,5-dione using N,N′-dicyclohexylcarbodiimide inTHF. Material is purified by prep HPLC.

General Procedure X3.

N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-valyl-N˜5˜-carbamoyl-N-[4-({[(2-carboxypropan-2-yl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamideis coupled to #50 following general procedure D in DMSO andacetonitrile. Fmoc is removed following general procedure A followed bycoupling with bis(pentafluorophenyl)3,3′-[ethane-1,2-diylbis(oxy)]dipropanoate using Hunig's base inacetonitrile. Material is purified by prep HPLC.

General Procedure X4.

N-[1-(9H-fluoren-9-yl)-3,5,12-trioxo-2,7,10-trioxa-4-azadodecan-12-yl]-2-methylalanineis coupled to #250 following general procedure D in acetonitrile. Fmocis removed following general procedure A followed by coupling withbis(pentafluorophenyl) 3,3′-[ethane-1,2-diylbis(oxy)]dipropanoate usingHunig's base in acetonitrile. Material is purified by prep HPLC.

General Procedure X5.

L-valyl-N˜5˜-carbamoyl-N-[4-(hydroxymethyl)phenyl]-L-ornithinamide iscoupled to N˜2˜-acetyl-N˜6˜-(tert-butoxycarbonyl)-L-lysine followinggeneral procedure D. This resulting residue is coupled withbis(4-nitrophenyl)carbonate with Hunig's base in DMF, followed bycoupling with the desired cytotoxic pentapeptide following generalprocedure E. Boc de-protection is then performed following generalprocedure B in acetonitrile. Residue is purified by prep HPLC.

In some instances minor alterations to reaction conditions were madesuch as but not limited to order of reagent and reactant addition and orthe amount of reagent or reactant which is indicated by the symbol *.Furthermore, these general procedures are provided as exemplary only andare non-limiting.

In addition to the General Procedures provided above, relevantdolastatin and auristatin references include the following: Petit et al.J. Am. Chem. Soc. 1989, 111, 5463; Petit et al. Anti-Cancer Drug Design1998, 13, 243 and references cited therein; Petit et al. J. Nat. Prod.2011, 74, 962; WO 96/33212; WO 95/09864; EP 0695758; WO 07/8848; WO01/18032; WO 09/48967; WO 09/48967; WO 09/117,531; WO 08/8603; U.S. Pat.No. 7,750,116; U.S. Pat. No. 5,985,837; and US 2005/9751; all of whichare hereby incorporated by reference in their entireties.

MS Analysis and Sample Preparation

Samples were prepped for LC-MS analysis by combining about 20 μL ofsample (approximately 1 mg/mL of ADC in PBS) with 20 μL of 20 mMdithiothreitol (DTT). After allowing the mixture to stand at roomtemperature for 5 minutes, the samples were analyzed according toprotocol 0.

The following calculation was performed in order to establish the totalloading (DAR) of the conjugate:Loading=2*[LC1/(LC1+LC0)]+2*[HCl/(HCO+HC1+HC2+HC3)]+4*[HC2/(HC0+HC1+HC2+HC3)]+6*[HC3/(HC0+HC1+HC2+HC3)]Where the indicated variables are the relative abundance of:LC0=unloaded light chain, LC1=single loaded light chain, HC0=unloadedheavy chain, HC1=single loaded heavy chain, HC2=double loaded heavychain, and HC3=triple loaded heavy chain.

LC-MS conditions used are Protocol F for retention time below one minuteand Protocol H for the remaining experiments unless otherwise indicated.

Preparation ofN-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide(#8)

Step 1 Synthesis ofbenzyl[(2S,35)-1-hydroxy-3-methylpentan-2-yl]methylcarbamate (#1)

To a solution of N-[(benzyloxy)carbonyl]-N-methyl-L-isoleucine (52.37 g,187.5 mmol, 1 eq.) in tetrahydrofuran (524 mL, 0.35 M) was addedborane-tetrahydrofuran complex (1 M in tetrahydrofuran, 375 mL, 375mmol, 2 eq.) slowly over 1 hour and the reaction was allowed to stir for18 hours at room temperature. The reaction was cooled to 0° C. and water(30 mL) was added over 30 minutes. The reaction mixture was diluted with1 M aqueous sodium carbonate solution (100 mL) and tert-butyl methylether (250 mL). The aqueous layer was back-extracted with tert-butylmethyl ether (100 mL). The combined organic layers were washed with 1 Maqueous sodium carbonate solution (100 mL), washed with brine (200 mL),dried over magnesium sulfate, filtered, and concentrated in vacuo toprovide #1 (48.44 g, 97% yield) as a pale yellow oil, which was used inthe next step without further purification. ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers: δ 7.26-7.41 (m, 5H), [5.06 (ABquartet, J_(AB)=12.9 Hz, Δν_(AB)=22.8 Hz) and 5.06 (AB quartet,J_(AB)=9.0 Hz, Δν_(AB)=9.0 Hz), total 2H], [4.65 (t, J=5.3 Hz) and 4.59(t, J=5.4 Hz), total 1H], 3.67-3.80 (m, 1H), 3.51-3.60 (m, 1H),3.41-3.51 (m, 1H), 2.75 and 2.71 (2 s, total 3H), 1.49-1.64 (br m, 1H),1.24-1.37 (br m, 1H), 0.90-1.02 (br m, 1H), 0.74-0.87 (m, 6H).

Step 2 Synthesis of benzylmethyl[(2S,35)-3-methyl-1-oxopentan-2-yl]carbamate (#2)

To a solution of #1 (8.27 g, 31.2 mmol, 1 eq.) in dimethyl sulfoxide(41.35 mL, 0.75 M), was added triethylamine (8.70 mL, 64.0 mmol, 2.05eq.) and the mixture was cooled to 0° C. Sulfur trioxide pyridinecomplex (10.18 g, 63.96 mmol, 2.05 eq.) was then added portion-wise,while keeping the internal temperature below 8° C. The reaction wasallowed to reach room temperature and was stirred for 18 hours. Thereaction was poured into water (100 mL) and tert-butyl methyl ether (100mL). The aqueous layer was back-extracted with tert-butyl methyl ether(50 mL) and the combined organic layers were washed with brine (100 mL),dried over magnesium sulfate, filtered, concentrated in vacuo andpurified by silica gel chromatography (Gradient: 10% to 60% ethylacetate in heptane) to provide #2 (7.14 g, 87%) as a colorless oil. ¹HNMR (400 MHz, DMSO-d₆), presumed to be a mixture of rotamers,characteristic signals: δ 9.61 (s, 1H), 7.26-7.42 (m, 5H), 5.01-5.13 (m,2H), 4.04-4.12 (m, 1H), 2.86 and 2.82 (2 s, total 3H), 1.94-2.11 (br m,1H), 1.26-1.42 (br m, 1H).

Step 3 Synthesis of tert-butyl(3R,4S,5S)-4-{[(benzyloxy)carbonyl](methyl)amino}-3-hydroxy-5-methylheptanoate(#3)

Lithium diisopropylamine was prepared by adding n-butyllithium (2.5 Msolution in tetrahydrofuran, 35.9 mL, 89.8 mmol, 1.4 eq.) to a solutionof diisopropylamine (13.8 mL, 96.3 mmol, 1.5 eq.) in tetrahydrofuran (50mL, 1.3 M) at −78° C. After 1 hour, tert-butyl acetate (15.7 mL, 116mmol, 1.8 eq.) was added drop-wise and the reaction mixture was stirredfor an additional 1.5 hours while being allowed to slowly warm to -20°C. The reaction mixture was recooled to −78° C. and a solution of thealdehyde #2 (16.9 g, 64.2 mmol, 1 eq.) in tetrahydrofuran (10 mL) wasadded. The reaction mixture was stirred for 1.5 hours and then quenchedby addition of water (100 mL). After extraction with diethyl ether(2×100 mL), the combined organic layers were dried over sodium sulfate,filtered, concentrated in vacuo and purified by silica gelchromatography (Gradient: 0% to 20% acetone in heptane) to provide #3(8.4 g, 34%) as a colorless oil. LC-MS: m/z 402.4 [M+Na⁺], retentiontime=3.91 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixtureof rotamers: δ 7.27-7.39 (m, 5H), 5.01-5.12 (m, 2H), [4.93 (d, J=7.2 Hz)and 4.98 (br d, J=7.2 Hz), total 1H], 4.03-4.15 (br m, 1H), 3.68-3.85(br m, 1H), 2.65 and 2.72 (2 br s, total 3H), 2.28-2.37 (m, 1H),2.09-2.17 (m, 1H), 1.74-1.90 (br m, 1H), 1.41-1.51 (m, 1H), 1.39 (s,9H), 0.92-1.01 (m, 1H), 0.77-0.92 (m, 6H).

Step 4 Synthesis of tert-butyl(3R,4S,5S)-4-{[(benzyloxy)carbonyl](methyl)amino}-3-methoxy-5-methylheptanoate(#@2)

To a solution of #3 (8.4 g, 22 mmol, 1 eq.) in 1,2-dichloroethane (25mL, 0.88 M) were added molecular sieves (4 Å, 0.7 g) and Proton sponge(1,8-bis(dimethylamino)naphthalene) (13.4 g, 59.2 mmol, 2.7 eq.),followed by trimethyloxonium tetrafluoroborate (9.10 g, 61.6 mmol, 2.8eq.). After stirring overnight, the reaction mixture was filteredthrough Celite. The filtrate was concentrated in vacuo and the residuewas purified by silica gel chromatography (Gradient: 0% to 40% 1:1acetone:ethyl acetate in heptane) to give #@2 (8.7 g, 68%) as acolorless oil. ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixture ofrotamers: δ 7.28-7.40 (m, 5H), 5.01-5.13 (m, 2H), 3.89-4.08 (br m, 1H),3.70-3.82 (m, 1H), 3.18 and 3.26 (2 s, total 3H), 2.66 and 2.71 (2 br s,total 3H), 2.44-2.53 (m, 1H, assumed; partially obscured by solventpeak), 2.17-2.24 (m, 1H), 1.71-1.86 (br m, 1H), 1.39 and 1.39 (2 s,total 9H), 1.31-1.40 (m, 1H), 0.94-1.08 (m, 1H), 0.76-0.91 (m, 6H).

Step 5 Synthesis of tert-butyl(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoate, hydrochloridesalt (#4)

To a solution of #@2 (13.37 g, 33.98 mmol, 1 eq.) in methanol (134 mL,0.1 M) and concentrated hydrochloric acid (3.1 mL, 37.4 mmol, 1.1 eq.)was added 10% palladium on carbon (50% wet) (0.1 wt %; 1.34 g, 3.40mmol). The mixture was hydrogenated at 45 psi for 3 hours, then purgedwith nitrogen, filtered through Celite and concentrated in vacuo toprovide #4 (9.20 g, 92%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ9.65 (br s, 1H), 8.97 (br s, 1H), 3.98-4.04 (m, 1H), 3.40 (s, 3H),3.06-3.13 (br m, 1H), 2.82 (br dd, J=6, 5 Hz, 3H), 2.74-2.80 (m, 1H),2.68 (dd, half of ABX pattern, J=16.3, 4.2 Hz, 1H), 2.00-2.10 (br m,1H), 1.73-1.84 (m, 1H), 1.46 (s, 9H), 1.38-1.45 (m, 1H), 1.13 (d, J=7.0Hz, 3H), 0.99 (t, J=7.4 Hz, 3H).

Step 6 Synthesis of tert-butyl(3R,4S,5S)-4-[{N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoate(#5)

To a mixture of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valine (18.53 g,54.60 mmol, 1.3 eq.) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT)(9.58 g, 54.6 mmol, 1.3 eq.) in 2-methyltetrahydrofuran (118.00 mL, 0.34M) was added N-methylmorpholine (6.52 mL, 59.1 mmol, 1.5 eq.) followedby #4 (11.80 g, 39.9 mmol, 1 eq.). After 3 hours, the reaction wasquenched with water (50 mL) and stirred for 15 minutes. The aqueouslayer was separated and back-extracted with 2-methyltetrahydrofuran (50mL). The combined organic layers were washed with saturated aqueoussodium bicarbonate solution (50 mL), dried over magnesium sulfate,filtered, and concentrated in vacuo to give a colorless oil, which waspurified by silica gel chromatography (Gradient: 5% to 40% ethyl acetatein heptane) to give #5 (26.2 g, 91%) as a colorless foam. LC-MS(Protocol I) m/z 581.3 [M+H⁺] 1604.3 [M+Na⁺], retention time=4.993minutes; ¹H NMR (400 MHz, DMSO-d₆), possibly a mixture of rotamers,characteristic major signals: δ 7.88 (d, J=7.4 Hz, 2H), 7.71 (d, J=7.4Hz, 2H), 7.62 (d, J=8.6 Hz, 1H), 7.41 (dd, J=7.4, 7.4 Hz, 2H), 7.27-7.34(m, 2H), 4.13-4.32 (m, 4H), 3.70-3.82 (br m, 1H), 3.24 (s, 3H), 2.92 (brs, 3H), 2.54 (dd, J=15.7, 2.4 Hz, 1H), 2.17 (dd, J=15.4, 9.4 Hz, 1H),1.95-2.07 (m, 1H), 1.70-1.83 (br m, 1H), 1.40 (s, 9H), 0.83-0.94 (m,9H), 0.69 (t, J=7.2 Hz, 3H).

Step 7A Synthesis of tert-butyl(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoate (#6)

To a solution of #5 (26 g, 42 mmol, 1 eq.) in tetrahydrofuran (260 mL,0.16 M) was added diethylamine (22 mL) over 30 minutes. The reaction wasstirred for about 6 hours and the suspension was then filtered throughCelite and washed with additional tetrahydrofuran (25 mL). The filtratewas concentrated in vacuo to provide a pale yellow oil, which wasredissolved in 2-methyltetrahydrofuran (50 mL) and concentrated again toensure complete removal of diethylamine. The crude oil of #6 (>15.25 g)was taken into the next step without further purification.

Step 7B Synthesis of(3R,4S,5S)-4-[{N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoicacid (#@5)

According to general procedure B, from #5 (1.62 g, 2.79 mmol, 1 eq.),dichloromethane (10 mL, 0.3 M) and trifluoroacetic acid (3 mL) wassynthesized #@5 (1.42 g, 97%) as a solid which was used without furtherpurification. LC-MS m/z 525.3 [M+H⁺] 1547.3 [M+Nal retention time=0.95minute; ¹H NMR (400 MHz, DMSO-d₆), characteristic signals: δ 7.89 (d,J=7.6 Hz, 2H), 7.71 (d, J=7.4 Hz, 2H), 7.59 (d, J=8.8 Hz, 1H), 7.41 (dd,J=7.6, 7.4 Hz, 2H), 7.28-7.34 (m, 2H), 4.14-4.32 (m, 4H), 3.24 (s, 3H),2.92 (br s, 3H), 2.51-2.57 (m, 1H, assumed; partially obscured bysolvent peak), 2.20 (dd, J=15.9, 9.5 Hz, 1H), 1.95-2.06 (m, 1H),1.70-1.83 (br m, 1H), 1.22-1.36 (br m, 1H), 0.84-0.93 (m, 9H), 0.70 (t,J=7.3 Hz, 3H).

Step 8 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N—[(3R,4S,55)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#7)

To a mixture of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valine(19.54 g, 55.29 mmol, 1.3 eq.) and #6 (15.25 g, 42.54 mmol, 1 eq.) in2-methyltetrahydrofuran (152 mL, 0.28 M) was added2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (9.71 g, 55.3 mmol, 1.3eq.). After 10 minutes, N-methylmorpholine (6.6 mL, 60 mmol, 1.4 eq.)was slowly added, while keeping the internal temp below 25° C. Thereaction was stirred for 4 hours and was then quenched by the additionof water (50 mL). After stirring for 15 minutes, the aqueous layer wasseparated and back-extracted with 2-methyltetrahydrofuran (50 mL). Thecombined organic layers were washed with saturated aqueous sodiumbicarbonate solution (100 mL), then were dried over magnesium sulfate,filtered, and concentrated in vacuo. The resulting yellow foam waspurified by silica gel chromatography (Gradient: 5% to 35% ethyl acetatein heptane) to give #7 (32 g, 97%). ¹H NMR (400 MHz, DMSO-d₆), presumedto be a mixture of rotamers, characteristic signals: δ 7.89 (d, J=7.4Hz, 2H), 7.62 (d, J=7.4 Hz, 2H), 7.41 (br dd, J=7.4, 7.4 Hz, 2H),7.29-7.34 (m, 2H), 4.52-4.69 (br m, 1H), 3.70-3.82 (br m, 1H), 3.22 and3.25 (2 br s, total 3H), 2.94 and 2.96 (2 br s, total 3H), 2.78 and 2.81(2 br s, total 3H), 2.11-2.23 (m, 1H), 1.90-2.10 (m, 2H), 1.68-1.83 (brm, 1H), 1.40 (s, 9H), 1.21-1.33 (br m, 1H).

Step 9 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide(#8)

To #7 (32 g, 46 mmol, 1 eq.) in dichloromethane (160 mL, 0.29 M) wasadded drop-wise over 10 minutes trifluoroacetic acid (17.4 mL, 231 mmol,5 eq.). After 6 hours, the same amount of trifluoroacetic acid was addedand the reaction was continued for 18 hours. The reaction mixture wasdiluted with toluene (320 mL) and concentrated in vacuo to provide #8(35.8 g, 97%) as a pinkish oil, which was used in the next step withoutfurther purification. ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ 7.90 (d, J=7.0 Hz, 2H),7.62 (d, J=7.4 Hz, 2H), 7.41 (br dd, J=7.4, 7.0 Hz, 2H), 7.29-7.35 (m,2H), 4.54-4.68 (br m, 1H), [4.09 (d, J=11 Hz) and 4.22 (d, J=10.9 Hz),total 1H], 3.74-3.84 (br m, 1H), 3.22 and 3.24 (2 br s, total 3H), 2.94and 2.96 (2 br s, total 3H), 2.78 and 2.80 (2 br s, total 3H), 2.13-2.24(m, 1H), 1.89-2.10 (br m, 2H), 1.70-1.81 (br m, 1H).

Preparation of(2R,3R)-3-[(2S)-1-(tert-Butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoicacid (#11; “Boc-Dap-acid”)

Step 1 Synthesis of tert-butyl(2S)-2-[(1R,2S)-1-hydroxy-2-methylbut-3-en-1-yl]pyrrolidine-1-carboxylate(#9)

To a solution of tert-butyl (2S)-2-formylpyrrolidine-1-carboxylate (10g, 50 mmol, 1 eq.) in dichloromethane (120 mL, 0.42 M) was addedpotassium (2Z)-2-buten-1-yltrifluoroborate (9.76 g, 60.2 mmol, 1.2 eq.)followed by tetra-n-butylammonium bromide (3.24 g, 5.02 mmol, 0.1 eq.)and water (60 mL). After 13 hours, the reaction was diluted withdichloromethane (150 mL) and water (150 mL). The aqueous layer wasseparated and back-extracted with dichloromethane (100 mL). The combinedorganic layers were washed with aqueous sodium chloride solution (5% wt,200 mL), washed with water (200 mL), and concentrated in vacuo to afford#9 (˜13 g) as an orange oil, which was used without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ 5.61-5.86 (br m, 1H), 4.97-5.09(m, 2H), 3.80-3.98 (br m, 2H), 3.45-3.67 (br m, 1H), 3.21-3.29 (m, 1H),2.14-2.26 (m, 1H), 1.80-2.04 (m, 3H), 1.65-1.76 (m, 1H), 1.47 (s, 9H),1.12 (d, J=6.6 Hz, 3H).

Step 2 Synthesis of tert-butyl(2S)-2-[(1R,2S)-1-methoxy-2-methylbut-3-en-1-yl]pyrrolidine-1-carboxylate(#10)

Sodium hydride (60% in mineral oil, 3.38 g, 84.4 mmol, 1.1 eq.) wascombined with hexane (40 mL), and the mixture was subjected to rapidmechanical stirring for 5 minutes. The solids were allowed to settle andthe hexane was removed. This procedure was repeated twice to removemineral oil. N,N-Dimethylformamide (59 mL, 1.3 M) was added and themixture was cooled to 0° C.; methyl iodide (5 mL; 81 mmol, 1.05 eq.) wasthen added drop-wise, followed by drop-wise addition of a solution of #9(19.6 g, 76.8 mmol, 1 eq.) in N,N-dimethylformamide (59 mL) over 5minutes, while keeping the temperature between 0° C. and 5° C. Thereaction was stirred at 0° C. for 2 hours. The reaction was quenchedwith saturated aqueous ammonium chloride solution (150 mL), poured intoaqueous sodium chloride solution (5% wt, 300 mL), and the mixture wasextracted with ethyl acetate (3×50 mL). The combined organic layers werewashed with 10% aqueous sodium chloride solution (2×300 mL), washed withwater (200 mL), and concentrated in vacuo. The resulting water-wet oilwas reconcentrated from ethyl acetate (150 mL) and purified by silicagel chromatography (Gradient: 2% to 10% ethyl acetate in heptane) toafford #10 (15.0 g, 73%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃),presumed to be a mixture of rotamers: δ 5.60-5.83 (m, 1H), 4.91-5.06 (m,2H), 3.81-3.95 (br m, 1H), 3.43 (s, 3H), 3.36-3.61 (m, 2H), 3.19-3.31(m, 1H), 2.09-2.21 (m, 1H), 1.86-2.02 (br m, 2H), 1.62-1.85 (br m, 2H),1.47 and 1.49 (2 s, total 9H), 1.09 (d, J=6.6 Hz, 3H).

Step 3 Synthesis of(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoicacid (#11)

To #10 (25.0 g, 92.8 mmol, 1 eq.) in tert-butanol (100 mL, 0.93 M) wasimmediately added water (30.00 mL) followed byN-methylmorpholine-N-oxide (25.97 g, 192.1 mmol, 2.07 eq.) and osmiumtetroxide (235.93 mg, 928.04 μmol, 0.01 eq.) After 12 hours, the mixturewas concentrated in vacuo using water (20 mL) to azeotropically removeresidual tert-butanol. The residue was partitioned between ethyl acetate(500 mL) and water (500 mL) plus brine (150 mL). The aqueous layer wasre-extracted with ethyl acetate (250 mL). The combined organic layerswere washed with aqueous sodium chloride solution (10 wt %, 200 mL),washed with water (150 mL), and concentrated in vacuo to afford awater-wet pale brown oil that was re-concentrated from ethyl acetate(100 mL) to remove any remaining water. This crude diol (34.76 g) wasused without further purification.

To the crude diol (34.76 g, ≦92.8 mmol, 1 eq.) in acetonitrile (347 mL,0.1 M) and water (174 mL) was added sodium permanganate (2.03 g, 5.73mmol, 0.05 eq.). The mixture was cooled to 0° C. and sodium periodate(51.46 g, 240.6 mmol, 2.1 eq.) was added portion-wise over 30 minutes,while keeping the internal temperature below 5° C. The reaction wasstirred at 0° C. for 4 hours and was then poured into a solution ofsodium thiosulfate pentahydrate (65.40 g, 263.5 mmol, 2.3 eq.) in water(100 mL). The mixture was filtered through Celite and the filtrate wasconcentrated in vacuo. The residue was partitioned between ethyl acetate(200 mL) and water (200 mL). The aqueous layer was back-extracted withethyl acetate (250 mL), and the combined organic layers were washed witha 10% aqueous citric acid solution. As the desired product was verysoluble in water, all the aqueous layers were combined, treated withCelite (100 g) and concentrated in vacuo to yield an off-white paste.Ethyl acetate (150 mL) was added and the mixture was re-concentrated toremove any residual water; this operation was repeated one more time.The paste was treated with ethyl acetate (150 mL) and placed in vacuo at50° C. for 10 minutes and filtered (repeated twice). These filtrateswere combined with the previous organic layer (from the citric acidwash), concentrated, diluted with ethyl acetate (200 mL) and filteredthrough Celite to remove solids. Finally, this filtrate was concentratedto yield #11 (22.9 g, 69% over two steps) as a yellowish/brown foam.LCMS (Protocol I): m/z 310.1 [M+Na⁺], 232.1 [(M-2-methylprop-1-ene)+H⁺], 188.1 [(M−Boc)+H⁺], retention time=3.268minutes; ¹H NMR (400 MHz, DMSO-d₆), characteristic signals: δ 3.61-3.85(br m, 2H), 3.20-3.45 (br m, 4H), 3.03-3.17 (br m, 1H), 1.59-1.93 (br m,4H), 1.40 (br s, 9H), 1.02-1.18 (br m, 3H).

Preparation of(2R,3R)-3-Methoxy-2-methyl-N-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]-3-[(2S)-pyrrolidin-2-yl]propanamide,trifluoroacetic acid salt (#19) and(2R,3R)-3-methoxy-2-methyl-N-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]-3-[(2S)-pyrrolidin-2-yl]propanethioamide,trifluoroacetic acid salt (#18)

Step 1 Synthesis of Nα-(tert-butoxycarbonyl)-L-phenylalaninamide (#12)

To a solution of Boc-Phe-OH (30.1 g, 113 mmol, 1 eq.) in tetrahydrofuran(378 mL, 0.3 M) cooled to −10° C. were added N-methylmorpholine (13.6mL, 124 mmol, 1.09 eq.), and ethyl chloroformate (11.8 mL, 124 mmol,1.09 eq.). After 20 minutes, a 30% aqueous ammonium hydroxide solution(45 mL, 350 mmol, 3.1 eq.) was added. The mixture was stirred at roomtemperature for 18 hours before being concentrated in vacuo. The residuewas diluted with ethyl acetate and washed sequentially with 1 N aqueouspotassium bisulfate solution, water and brine. The organic layer wasthen dried over sodium sulfate, filtered, and concentrated in vacuo. Thewhite solid was dissolved (this required heating with stirring) in ethylacetate (about 400 mL); the solution was then allowed to cool to roomtemperature before adding hexane (1000 mL). After a few minutes, a whitematerial started to precipitate from the reaction mixture. The solid wascollected by filtration, washed with heptane (2×˜150 mL), and driedunder vacuum for 18 hours to give #12 (24.50 g, 82%) as a solid. LC-MS:m/z 263.2 [M−H⁺], 309.2 [M+HCO₂ ⁻], retention time=1.85 minutes; ¹H NMR(400 MHz, DMSO-d₆), presumed to be a mixture of rotamers, major rotamer:δ 7.35 (br s, 1H), 7.22-7.30 (m, 5H), 7.00 (br s, 1H), 6.78 (d, J=8.6Hz, 1H), 4.09 (ddd, J=10, 9, 4.5 Hz, 1H), 2.95 (dd, J=13.8, 4.4 Hz, 1H),2.72 (dd, J=13.7, 10.1 Hz, 1H), 1.30 (s, 9H).

Step 2 Synthesis of tert-butyl[(2S)-1-amino-3-phenyl-1-thioxopropan-2-yl]carbamate (#13)

To a solution of #12 (14.060 g, 53.192 mmol, 1 eq.) in tetrahydrofuran(180 mL, 0.296 M), was added2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-dithione(Lawesson's reagent) (12.70 g, 31.40 mmol, 0.59 eq.) and the reactionwas refluxed for 90 minutes. The reaction was cooled to room temperatureand quenched by addition of saturated aqueous sodium bicarbonatesolution. The mixture was extracted twice with ethyl acetate and thecombined organic layers were dried over sodium sulfate, filtered, andconcentrated in vacuo. The residue was dissolved in ethyl acetate,concentrated in vacuo onto silica and purified by silica gelchromatography (Gradient: 0% to 100% ethyl acetate in heptane),affording #13 (11.50 g, 77%) as a white solid. LC-MS: m/z 279.4 [M−H⁺],225.2 [(M−2-methylprop-1-ene)+H⁺], 181.2 [(M−Boc)+H⁺]; ¹H NMR (400 MHz,DMSO-d₆), presumed to be a mixture of rotamers, major rotamer: δ 9.60(br s, 1H), 9.19 (br s, 1H), 7.23-7.32 (m, 5H), 6.82 (d, J=8.8 Hz, 1H),4.44 (ddd, J=9.4, 9.1, 4.4 Hz, 1H), 3.00 (dd, J=13.7, 4.5 Hz, 1H), 2.79(dd, J=13.6, 9.9 Hz, 1H), 1.29 (s, 9H).

Step 3 Synthesis of tert-butyl[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]carbamate (#14)

To a mixture of #13 (5.65 g, 20.2 mmol, 1 eq.) in acetone (101 mL, 0.2M) was added bromoacetaldehyde diethyl acetal (8.76 mL, 58.2 mmol, 2.89eq.) and 2 drops of 4 M hydrochloric acid in dioxane. The mixture wasdegassed with nitrogen three times before being heated to reflux. After2 hours, the reaction was cooled to room temperature and concentrated invacuo. The residue was dissolved in ethyl acetate, washed with saturatedaqueous sodium bicarbonate solution and washed with brine. The organiclayer was dried over sodium sulfate, filtered, and concentrated invacuo. The resulting crude orange oil was diluted with ethyl acetatebefore being concentrated in vacuo onto silica and purified by silicagel chromatography (Gradient: 0% to 35% ethyl acetate in heptane) andthen by reverse phase chromatography (Method A) to give #14 (625 mg,10%); HPLC (Protocol E): m/z 304.5 [M+H⁺], 248.9[(M−2-methylprop-1-ene)+H⁺], retention time=7.416 minutes; ¹H NMR (400MHz, DMSO-d₆), presumed to be a mixture of rotamers, major rotamer: δ7.75 (d, J=3.3 Hz, 1H), 7.75 (br d, J=8.6 Hz, 1H), 7.61 (br d, J=3.1 Hz,1H), 7.25-7.30 (m, 5H), 4.99 (ddd, J=10.5, 8.9, 4.5 Hz, 1H), 3.29-3.36(m, 1H, assumed; partially obscured by water signal), 2.98 (dd, J=13.8,10.6 Hz, 1H), 1.31 (s, 9H).

Step 4 Synthesis of (1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethanamine,hydrochloride salt (#15)

According to general procedure C, from #14 (1.010 g, 3.318 mmol, 1 eq.),dioxane (10 mL, 0.33 M) and a 4 M solution of hydrochloric acid indioxane (20 mL, 80 mmol, 20 eq.) was synthesized #15 (775 mg, 97%). ¹HNMR (400 MHz, DMSO-d₆) δ 8.95-9.07 (br m, 3H), 7.86 (d, J=3.2 Hz, 1H),7.73 (d, J=3.2 Hz, 1H), 7.18-7.28 (m, 3H), 7.10-7.15 (m, 2H), 4.98-5.07(m, 1H), 3.49 (dd, J=13.3, 4.9 Hz, 1H), 3.18 (dd, J=13.4, 10.2 Hz, 1H).

Step 5 Synthesis of tert-butyl(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidine-1-carboxylate(#16)

To a solution of #11 (280 mg, 0.974 mmol, 1 eq.) and #15 (460 mg, 1.44mmol, 1.48 eq.) in N,N-dimethylformamide (3 mL, 0.32 M) at 0° C. wasadded diethylphosphoryl cyanide (DEPC) (93% purity, 212 μL, 1.30 mmol,1.34 eq.), followed by triethylamine (367 μL, 2.63 mmol, 2.7 eq.). After2 hours at 0° C., the reaction mixture was warmed to room temperaturefor 18 hours. The reaction mixture was then diluted with ethylacetate:toluene (2:1, 30 mL) and was washed successively with 1 Maqueous sodium bisulfate solution (35 mL) and 50% saturated aqueoussodium bicarbonate solution (4×25 mL). The organic layer was dried oversodium sulfate, filtered, concentrated in vacuo, and purified by silicagel chromatography (12% to 100% ethyl acetate in heptane) to give #16 asa light amber oil (374 mg, 81%). LC-MS: m/z 474.4 [M+H⁺], 374.4[(M−2-methylprop-1-ene)+H⁺] retention time=3.63 minutes; ¹H NMR (400MHz, DMSO-d₆), characteristic signals: δ 8.66 (d, J=8.5 Hz, 1H), 7.78(d, J=3.3 Hz, 1H), 7.64 (d, J=3.3 Hz, 1H), 7.21-7.31 (m, 4H), 7.14-7.20(m, 1H), 5.40 (ddd, J=11.4, 8.5, 4.0 Hz, 1H), 3.23 (br s, 3H), 2.18 (dq,J=9.7, 6.7 Hz, 1H), 1.06 (d, J=6.6 Hz, 3H).

Step 6A Synthesis of tert-butyl(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidine-1-carboxylate(#17)

A mixture of #16 (350 mg, 0.739 mmol, 1 eq.) and2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-dithione(Lawesson's reagent) (324 mg, 0.776 mmol, 1.05 eq.) in toluene (6 mL,0.1 M) was warmed to 100° C. After 10 minutes, the mixture was cooled toroom temperature. Insoluble material was removed by filtration and thefiltrate was concentrated in vacuo. The residue was purified by silicagel chromatography (Gradient: 12% to 80% ethyl acetate in heptane) andthen by reverse phase chromatography (Method E2) to give #17 (120 mg,33%); HPLC (Protocol J): m/z 490.2 [M+H⁺], retention time=10.069minutes; [α]²⁰ _(D)−110 (c 0.24, MeOH); ¹H NMR (400 MHz, CD₃OD),characteristic signals: δ 7.78 (d, J=3.3 Hz, 1H), 7.51 (d, J=3.3 Hz,1H), 7.32-7.37 (m, 2H), 7.24-7.30 (m, 2H), 7.17-7.23 (m, 1H), 6.52-6.61(br m, 1H), 3.62 (br dd, J=15, 4 Hz, 1H), 3.37 (s, 3H), 2.98-3.09 (br m,1H), 2.53-2.64 (br m, 1H), 1.60-1.78 (m, 2H), 1.49 (s, 9H), 1.27 (d,J=6.5 Hz, 3H).

Step 6B Synthesis of(2R,3R)-3-methoxy-2-methyl-N-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]-3-[(2S)-pyrrolidin-2-yl]propanethioamide,trifluoroacetic acid salt (#18)

According to general procedure B, from #17 (198 mg, 0.404 mmol, 1 eq.),dichloromethane (6 mL, 0.07 M) and trifluoroacetic acid (2 mL) wassynthesized #18 (185 mg, 91%), which was used without furtherpurification. LC-MS: m/z 390.1 [M+H⁺], retention time=0.57 minutes; ¹HNMR (400 MHz, DMSO-d₆) δ 10.91 (d, J=8.2 Hz, 1H), 9.07-9.20 (br m, 1H),7.86-8.00 (br m, 1H), 7.83 (d, J=3.2 Hz, 1H), 7.69 (d, J=3.3 Hz, 1H),7.27-7.36 (m, 4H), 7.21-7.26 (m, 1H), 6.33 (ddd, J=11.3, 8.3, 4.4 Hz,1H), 3.76-3.82 (m, 1H), 3.56 (dd, J=14.6, 4.3 Hz, 1H), 3.45 (s, 3H),3.28 (dd, J=14.6, 11.3 Hz, 1H), 3.02-3.12 (br m, 1H), 2.89-3.00 (br m,1H), 2.72-2.89 (m, 2H), 1.69-1.83 (br m, 1H), 1.43-1.58 (m, 2H),1.20-1.33 (m, 1H), 1.22 (d, J=6.6 Hz, 3H).

Step 7 Synthesis of(2R,3R)-3-methoxy-2-methyl-N-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]-3-[(2S)-pyrrolidin-2-yl]propanamide,trifluoroacetic acid salt (#19)

According to general procedure B, from #16 (607 mg, 1.28 mmol, 1 eq.),dichloromethane (10 mL, 0.13 M) and trifluoroacetic acid (2 mL) wassynthesized #19 (640 mg, quantitative), which was used in the next stepwithout further purification. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96-9.07 (brm, 1H), 8.89 (d, J=8.8 Hz, 1H), 7.87-8.00 (br m, 1H), 7.80 (d, J=3.2 Hz,1H), 7.66 (d, J=3.3 Hz, 1H), 7.28-7.34 (m, 4H), 7.20-7.27 (m, 1H), 5.43(ddd, J=11.3, 8.6, 4.2 Hz, 1H), 3.42-3.50 (m, 2H), 3.36 (s, 3H),3.04-3.14 (br m, 1H), 2.99 (dd, J=14.2, 11.5 Hz, 1H), 2.92-3.02 (m, 1H),2.78-2.88 (br m, 1H), 2.34-2.42 (m, 1H), 1.73-1.84 (br m, 1H), 1.55-1.68(m, 1H), 1.38-1.53 (m, 2H), 1.15 (d, J=6.9 Hz, 3H).

Preparation of(2R,3R)-3-Methoxy-2-methyl-N-(2-phenylethyl)-3-[(2S)-pyrrolidin-2-yl]propanethioamide,hydrochloride salt (#23) and(2R,3R)-3-methoxy-2-methyl-N-(2-phenylethyl)-3-[(2S)-pyrrolidin-2-yl]propanamide,hydrochloride salt (#24)

Step 1A Synthesis of tert-butyl(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidine-1-carboxylate(#20)

To #11 (22 g, 77 mmol, 1 eq.) in dichloromethane (383 mL, 0.2 M) andN,N-dimethylformamide (30 mL) were added diisopropylethylamine (26.9 mL,153 mmol, 2 eq.), 2-phenylethylamine (11.6 mL, 91.9 mmol, 1.2 eq.) andHATU (39.0 g, 99.5 mmol, 1.3 eq.). The reaction was stirred for 18 hoursand then concentrated in vacuo. The residue was taken up in ethylacetate (700 mL) and washed sequentially with 1 M aqueous hydrochloricacid solution (2×200 mL) and brine. The organic layer was dried oversodium sulfate, filtered and evaporated in vacuo. The crude material wastaken up in dichloromethane and filtered. The filtrate was purified bysilica gel chromatography (Gradient: 0% to 100% ethyl acetate inheptane) to give #20 (24 g, 80%) as an off-white solid. LC-MS: m/z 392.2[M+2H⁺], 291.1 [(M−Boc)+H⁺], retention time=0.88 minutes; ¹H NMR (400MHz, DMSO-d₆), presumed to be a mixture of rotamers: δ 7.80-7.89 (br m,1H), 7.23-7.29 (m, 2H), 7.15-7.23 (m, 3H), 3.72-3.82 and 3.55-3.62 (2 brm, total 1H), 3.45-3.55 (br m, 1H), 3.31-3.44 (br m, 2H), 3.29 (s, 3H),3.12-3.25 (br m, 1H), 2.98-3.12 (br m, 1H), 2.71 (t, J=7.1 Hz, 2H),2.09-2.19 (m, 1H), 1.71-1.83 (br m, 2H), 1.60-1.70 (br m, 1H), 1.49-1.60(br m, 1H), 1.41 (s, 9H), 1.03 (d, J=6.8 Hz, 3H).

Step 1B Synthesis of dipyridinium-1-ylpentathiodiphosphonate (#21)

Phosphorous pentasulfide (4.45 g, 2.19 mL, 20 mmol, 1 eq.) was added topyridine (56 mL, 0.36 M) at 80° C. and the mixture was heated at reflux(115° C.) for 1 hour. The mixture was cooled to room temperature and theproduct was collected by filtration to give #21 as a yellow solid (4.57g, 60%); mp: 165-167° C. (decomposition); ¹H NMR (400 MHz, DMSO-d₆) δ8.78-8.84 (m, 4H), 8.22-8.30 (m, 2H), 7.76-7.83 (m, 4H).

Step 2A Synthesis of tert-butyl(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2-phenylethyl)amino]-3-thioxopropyl}pyrrolidine-1-carboxylate(#22)

A mixture of #20 (1.200 g, 3.073 mmol, 1 eq.) and #21 (1.40 g, 3.69mmol, 1.2 eq.) in acetonitrile (15 mL, 0.20 M) was subjected tomicrowave radiation at 100° C. for 30 minutes. The reaction mixture wasthen cooled to room temperature, diluted with ethyl acetate (150 mL),and washed sequentially with 0.5 M aqueous hydrochloric acid solution(100 mL) and brine (2×50 mL). The organic layer was dried over magnesiumsulfate, filtered, and concentrated in vacuo. The residue was purifiedby silica gel chromatography (Gradient: 20% to 80% ethyl acetate inheptane) to give #22 (670 mg, 54%) as a white wax-like solid; mp:107-109° C.; LC-MS: m/z 407.4 [M+H⁺], 351.3 [(M−2-methylprop-1-ene)+H⁺],307.3 [(M−Boc)+H⁺], retention time=0.99 minutes; ¹H NMR (400 MHz,CD₃CN), presumed to be a mixture of rotamers: δ 8.28 (br s, 1H),7.19-7.33 (m, 5H), 3.81-4.05 (br m, 2H), 3.60-3.81 (br m, 2H), 3.38-3.51(br m, 1H), 3.36 (s, 3H), 3.02-3.17 (br m, 1H), 2.89-3.02 (m, 2H),2.50-2.62 (br m, 1H), 1.71-1.85 (br m, 2H), 1.53-1.66 (br m, 2H), 1.45(br s, 9H), 1.23 (d, J=6.7 Hz, 3H).

Step 3 Synthesis of(2R,3R)-3-methoxy-2-methyl-N-(2-phenylethyl)-3-[(2S)-pyrrolidin-2-yl]propanethioamide,hydrochloride salt (#23)

According to procedure C, from #22 (325 mg, 0.799 mmol, 1 eq.), dioxane(5 mL, 0.2 M) and a 4 M hydrochloric acid solution in dioxane (4 mL, 16mmol, 20 eq.) was synthesized #23 (274 mg, quantitative) as a whitefoam; LC-MS: 308.2 [M+H⁺], retention time=0.55 minutes.

Step 2B Synthesis of(2R,3R)-3-methoxy-2-methyl-N-(2-phenylethyl)-3-[(2S)-pyrrolidin-2-yl]propanamide,hydrochloride salt (#24)

To #20 (7.00 g, 17.9 mmol, 1 eq.) in dioxane (50 mL, 0.36 M) andmethanol (2 mL) was added a 4 M solution of hydrochloric acid in dioxane(20 mL, 80 mmol, 4.4 eq.). After stirring for 18 hours, the mixture wasconcentrated to afford #24 (5.86 g, quantitative) as a gum, which wasused without further purification; LC-MS: 292.2 [M+H⁺], retentiontime=0.47 minutes.

Preparation ofN-Methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#26)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#25)

According to general procedure D, from #8 (480 mg, 0.753 mmol, 1 eq.),dichloromethane (10 mL, 0.07 M), N,N-dimethylformamide (2 mL), the amine#18 (401 mg, 0.941 mmol, 1.25 eq.), HATU (372 mg, 0.979 mmol, 1.3 eq.)and triethylamine (367 μL, 2.64 mmol, 3.5 eq.) was synthesized the crudedesired material, which was purified by silica gel chromatography(Gradient: 0% to 30% acetone in heptane) to afford #25 (711 mg, 75%) asa solid. LC-MS: m/z 1009.7 [M+H⁺], retention time=1.15 minutes; HPLC(Protocol B): m/z 505.3 [M+2H⁺]/2, retention time=10.138 minutes; ¹H NMR(400 MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ [10.54 (br d, J=8 Hz) and 10.81 (br d, J=8 Hz), total 1H],7.89 (br d, J=7 Hz, 2H), [7.80 (d, J=3.3 Hz) and 7.83 (d, J=3.2 Hz),total 1H], [7.64 (d, J=3.2 Hz) and 7.69 (d, J=3.2 Hz), total 1H], 7.62(br d, J=7 Hz, 2H), 7.37-7.44 (m, 2H), 7.28-7.35 (m, 4H), 7.20-7.27 (m,2H), 7.12-7.18 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, total 1H), [1.14(d, J=6.4 Hz) and 1.17 (d, J=6.3 Hz), total 3H].

Step 2 Synthesis ofN-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#26)

According to general procedure A, from #25 (701 mg, 0.694 mmol) indichloromethane (10 mL, 0.07 M) and diethylamine (10 mL) was synthesizedthe crude desired material, which was purified by silica gelchromatography (Gradient: 0% to 10% methanol in dichloromethane) to givea glass-like solid. Diethyl ether and heptane were added and the mixturewas concentrated in vacuo, producing #26 (501 mg, 92%) as a white solid.HPLC (Protocol A): m/z 787.4 [M+H⁺], retention time=7.229 minutes,(purity >97%); ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixture ofrotamers, characteristic signals: δ [10.54 (br d, J=8 Hz) and 10.81 (brd, J=8. Hz), total 1H], [7.99 (br d, J=9 Hz) and 8.00 (br d, J=9 Hz),total 1H], [7.80 (d, J=3.3 Hz) and 7.83 (d, J=3.3 Hz), total 1H], [7.65(d, J=3.2 Hz) and 7.69 (d, J=3.3 Hz), total 1H], 7.29-7.34 (m, 2H),7.19-7.28 (m, 2H), 7.13-7.19 (m, 1H), [6.31 (ddd, J=11, 8, 4.5 Hz) and6.45 (ddd, J=11.5, 8, 4.5 Hz), total 1H], [4.57 (dd, J=8.9, 8.7 Hz) and4.63 (dd, J=8.7, 8.7 Hz), total 1H], 3.16, 3.21, 3.24 and 3.25 (4 s,total 6H), 2.96 and 3.03 (2 br s, total 3H), [1.14 (d, J=6.6 Hz) and1.17 (d, J=6.4 Hz), total 3H].

Preparation ofN²-[(1-Anainocyclopentyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#30)

Step 1 Synthesis of tert-butyl(3R,4S,55)-4-[{N-[(1-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}cyclopentyl)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoate(#27)

To #6 (287 mg, 0.801 mmol, 1 eq.) in dichloromethane (4 mL, 0.2 M) wereadded 1-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}cyclopentanecarboxylicacid (309 mg, 0.879 mmol, 1.1 eq.), diisopropylethylamine (281 μL, 1.60mmol, 2 eq.) and HATU (376 mg, 0.960 mmol, 1.2 eq.). The mixture wasstirred for 18 hours and diluted with ethyl acetate (15 mL). Thereaction mixture was washed with 1 M aqueous hydrochloric acid solution(2×5 mL) and with brine (5 mL). The organic layer was dried over sodiumsulfate, filtered, and concentrated in vacuo. The crude material waspurified by silica gel chromatography (Gradient: 0% to 60% ethyl acetatein heptane) to provide #27 (502 mg, 91%) as a white foam. LC-MS: m/z692.3 [M+H⁺], 714.3 [M+Na⁺], 636.3 [(M−2-methylprop-1-ene)+H⁺],retention time=1.13 minutes; ¹H NMR (400 MHz, DMSO-d₆), characteristicsignals: δ 7.89 (br d, J=7.4 Hz, 2H), 7.67-7.75 (m, 2H), 7.60 (br s,1H), 7.38-7.44 (m, 2H), 7.30-7.36 (m, 2H), 7.21 (br d, J=8.8 Hz, 1H),4.44-4.59 (m, 2H), 4.17-4.27 (m, 3H), 3.68-3.78 (br m, 1H), 3.21 (s,3H), 2.88 (br s, 3H), 2.09-2.20 (m, 2H), 1.39 (s, 9H).

Step 2 Synthesis of(3R,4S,5S)-4-[{N-[(1—{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}cyclopentyl)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoicacid (#28)

To a solution of #27 (500 mg, 0.723 mmol) in dichloromethane (7 mL, 0.1M) was added trifluoroacetic acid (3 mL). The reaction mixture initiallybecame orange, then darkened over time. After stirring for 18 hours, thesolvent was removed in vacuo to give #28 (460 mg, quantitative) as adark brown glass, which was used without further purification. LC-MS:m/z 636.3 [M+H⁺].

Step 3 Synthesis of N²-[(1-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}cyclopentyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#29)

According to general procedure D, from #28 (50 mg, 0.079 mmol, 1 eq.)dichloromethane (3 mL, 0.03 M), N,N-dimethylformamide (0.5 mL), amine#18 (44 mg, 0.087 mmol, 1.1 eq.), triethylamine (33.0 μL, 0.237 mmol, 3eq.) and HATU (36 mg, 0.95 mmol, 1.2 eq.) was synthesized the crudedesired material, which was purified by silica gel chromatography(Gradient: 0% to 30% acetone in heptane) to give #29 (59 mg, 67%) as asolid. LC-MS: m/z 1007.5 [M+H⁺], retention time=1.11 minutes; ¹H NMR(400 MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ [10.54 (br d, J=8 Hz) and 10.80 (br d, J=8 Hz), total 1H],7.89 (br d, J=7 Hz, 2H), [7.80 (d, J=3.3 Hz) and 7.82 (d, J=3.1 Hz),total 1H], 7.68-7.75 (m, 2H), [7.64 (d, J=3.2 Hz) and 7.68 (d, J=3.2Hz), total 1H], 7.38-7.44 (m, 2H), 7.27-7.36 (m, 4H), 7.12-7.25 (m, 4H),[6.30 (ddd, J=11, 8, 4.5 Hz) and 6.39-6.48 (m), total 1H], [4.50 (br dd,J=8, 8 Hz) and 4.54-4.59 (m), total 1H], 4.17-4.29 (m, 3H), 2.89 and2.96 (2 br s, total 3H), [1.13 (d, J=6.5 Hz) and 1.16 (d, J=6.4 Hz),total 3H].

Step 4 Synthesis ofN²-[(1-aminocyclopentyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#30)

According to general procedure A, from #29 (54 mg, 0.054 mmol) indichloromethane (6 mL, 0.9 mM) and diethylamine (4 mL) was synthesizedthe crude desired material, which was purified by silica gelchromatography (Gradient: 0% to 10% methanol in dichloromethane) to giveexample #30 (26 mg, 61%) as a solid. HPLC (Protocol A): retentiontime=7.233 minutes, m/z 785.4 [M+H⁺], (purity >72%). ¹H NMR (400 MHz,DMSO-d₆), presumed to be a mixture of rotamers, characteristic signals:δ [10.54 (br d, J=8 Hz) and 10.82 (br d, J=8 Hz), total 1H], 8.19-8.27(m, 1H), [7.80 (d, J=3.2 Hz) and 7.83 (d, J=3.2 Hz), total 1H], [7.65(d, J=3.3 Hz) and 7.69 (d, J=3.3 Hz), total 1H], 7.28-7.33 (m, 2H),7.20-7.27 (m, 2H), 7.14-7.19 (m, 1H), [6.31 (ddd, J=11, 8, 4.5 Hz) and6.44 (ddd, J=11, 8, 4 Hz), total 1H], [4.53 (dd, J=9, 8 Hz) and 4.60(dd, J=9, 7.5 Hz), total 1H], 3.24 and 3.25 (2 s, total 3H), 3.17 and3.21 (2 s, total 3H), 2.93 and 3.00 (2 br s, total 3H), [1.14 (d, J=6.5Hz) and 1.17 (d, J=6.5 Hz), total 3H].

Preparation of2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#34)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#31)

To a solution of #6 (70% pure, 3.13 g, 6.1 mmol, 1 eq.) indichloromethane (40 mL, 0.15 M) were addedN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanine (1.99 g, 6.12 mmol,1 eq.), diisopropylethylamine (2.67 mL, 15.3 mmol, 2.5 eq.) and HATU(2.79 g, 7.35 mmol, 1.2 eq.). The reaction mixture was stirred for 18hours, diluted with ethyl acetate, washed with 1 M aqueous hydrochloricacid solution and washed with brine. The organic layer was dried oversodium sulfate, filtered, and concentrated in vacuo onto silica. Thematerial was then purified by silica gel chromatography (Gradient: 0% to45% ethyl acetate in heptane) to provide #31 (3.65 g, 90%) as a solid.LC-MS: m/z 665.5 [M+H⁺], 688.5 [M+Na⁺], 610.5[(M−2-methylprop-1-ene)+H⁺]; HPLC (Protocol C): retention time=9.455(purity >94%); ¹H NMR (400 MHz, DMSO-d₆), characteristic signals: δ 7.89(d, J=7.4 Hz, 2H), 7.67-7.74 (m, 2H), 7.39-7.48 (m, 3H), 7.31-7.36 (m,2H), 7.29 (br d, J=8.8 Hz, 1H), 4.47-4.60 (br m, 1H), 4.47 (dd, J=8.6,8.0 Hz, 1H), 4.18-4.28 (m, 3H), 3.69-3.79 (br m, 1H), 3.21 (s, 3H), 2.88(br s, 3H), 2.15 (dd, J=15.5, 9.3 Hz, 1H), 1.91-2.01 (m, 1H), 1.67-1.81(br m, 1H), 1.39 (s, 9H), 1.36 (br s, 3H), 1.30 (s, 3H), 0.75 (d, J=6.6Hz, 3H), 0.66-0.73 (br m, 3H).

Step 2 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide(#32)

According to general procedure B, from #31 (500 mg, 0.751 mmol) indichloromethane (7 mL, 0.1 M) and trifluoroacetic acid (3 mL) wassynthesized #32 as a glass (458 mg, quantitative), which was used in thenext step without further purification. LC-MS: m/z 611.4 [M+2H⁺], 632.2[M+Na⁺], retention time=0.94 minute.

Step 3 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#33)

According to general procedure D, from #32 (53.0 mg, ≦0.083 mmol, 1eq.), dichloromethane (4 mL, 0.02 M), N,N-dimethylformamide (1 mL),amine #18 (43.8 mg, 0.0870 mmol, 1 eq.), triethylamine (36 μL, 0.26mmol, 3 eq.) and HATU (39.5 mg, 0.104 mmol, 1.2 eq.) was synthesized thecrude desired material, which was purified by silica gel chromatography(Gradient: 0% to 30% acetone in heptane) to give #33 (60 mg, 69% overtwo steps). LC-MS: m/z 981.4 [M+H⁺], retention time=1.090 minutes; ¹HNMR (400 MHz, DMSO-d₆), presumed to be a mixture of rotamers,characteristic signals: δ [10.54 (br d, J=8 Hz) and 10.80 (br d, J=8Hz), total 1H], 7.86-7.91 (m, 2H), [7.80 (d, J=3.3 Hz) and 7.82 (d,J=3.3 Hz), total 1H], 7.68-7.74 (m, 2H), [7.64 (d, J=3.2 Hz) and 7.68(d, J=3.3 Hz), total 1H], 7.38-7.44 (m, 2H), 7.20-7.36 (m, 6H),7.12-7.17 (m, 1H), 6.27-6.34 and 6.40-6.47 (2 m, total 1H), 3.22 and3.24 (2 s, total 3H), 3.14 and 3.18 (2 s, total 3H), 2.90 and 2.97 (2 brs, total 3H), 1.37 (br s, 3H), 1.31 (2 br s, total 3H), [1.13 (d, J=6.6Hz) and 1.16 (d, J=6.5 Hz), total 3H].

Step 4 Synthesis of2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#34)

According to general procedure A, from #33 (55 mg, 0.055 mmol, 1 eq.) indichloromethane (6 mL, 0.009 M) and diethylamine (4 mL) was synthesizedthe crude desired material, which was purified by silica gelchromatography (Gradient: 0% to 5% methanol in dichloromethane) to give#34 (25 mg, 60%) as a solid. HPLC (Protocol A): m/z 759.4 [M+H⁺],retention time=7.088 minutes, (purity >75%). ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ [10.54(br d, J=8 Hz) and 10.81 (br d, J=8 Hz), total 1H], 8.01-8.08 (m, 1H),[7.80 (d, J=3.1 Hz) and 7.83 (d, J=3.3 Hz), total 1H], [7.65 (d, J=3.2Hz) and 7.69 (d, J=3.2 Hz), total 1H], 7.29-7.33 (m, 2H), 7.20-7.27 (m,2H), 7.13-7.19 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, total 1H), [4.49(dd, J=9, 8 Hz) and 4.56 (dd, J=9, 8 Hz), total 1H], 3.24 and 3.25 (2 s,total 3H), 3.17 and 3.21 (2 s, total 3H), 2.92 and 2.99 (2 br s, total3H), 1.20 and 1.21 (2 s, total 3H), 1.12 and 1.13 (2 s, total 3H),0.75-0.81 (m, 3H).

Preparation ofN-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2-phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#36)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2-phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#35)

To a mixture of #23 (337 mg, 0.983 mmol, 1 eq.) in dichloromethane (8mL, 0.1 M) and N,N-dimethylformamide (1 mL) were added #8 (564 mg, 0.885mmol, 0.9 eq.), diisopropylethylamine (383 mg, 2.95 mmol, 3 eq.) andHATU (472 mg, 1.18 mmol, 1.2 eq.). After 2 hours, the mixture wasdiluted with dichloromethane, washed sequentially with 0.1 M aqueoushydrochloric acid and with brine, dried over magnesium sulfate,filtered, and concentrated in vacuo. The residue was purified by reversephase chromatography (Method G) to give #35 (600 mg, 66%); LC-MS: m/z926.6 [M+H⁺], retention time=1.16 minutes; ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ [9.94(br t, J=5 Hz) and 10.16-10.23 (br m), total 1H], 7.90 (d, J=7.2 Hz,2H), [7.71 (br d, J=7 Hz) and 8.06 (br d, J=8 Hz), total 1H], 7.60-7.65(m, 2H), 7.41 (br dd, J=7, 7 Hz, 2H), 7.15-7.36 (m, 7H), 3.29 (s, 3H),1.16-1.22 (m, 3H).

Step 2 Synthesis ofN-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2-phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#36)

According to general procedure A, from #35 (465 mg, 0.502 mmol, 1 eq.)in dichloromethane (5 mL, 0.1 M) and diethylamine (5 mL) was synthesizedthe crude desired material, which was purified by silica gelchromatography (Gradient: 0% to 10% methanol in dichloromethane) to give#36 (310 mg, 88%) as a solid. LC-MS: m/z 704.6 [M+H⁺], retentiontime=0.74 minutes; HRMS: m/z calculated for C₃₈H₆₆N₅O₅S: 704.4779.found: 704.477 [M+H⁺]; ¹H NMR (400 MHz, CD₃OD), presumed to be a mixtureof rotamers, characteristic signals: δ 7.23-7.30 (m, 4H), 7.15-7.22 (m,1H), [4.68 (d, J=8.6 Hz) and 4.74 (d, J=8.0 Hz), total 1H], 3.39 and3.40 (2 s, total 3H), 3.12 and 3.22 (2 br s, total 3H), [2.82 (d, J=6.0Hz) and 2.84 (d, J=6.0 Hz), total 1H], 2.29 and 2.30 (2 s, total 3H),[1.27 (d, J=6.8 Hz) and 1.29 (d, J=6.6 Hz), total 3H], [0.84 (t, J=7.4Hz) and 0.87 (t, J=7.4 Hz), total 3H].

Preparation ofN-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#41) andN-Methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#42)

Step 1 Synthesis of methylN-{(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate(#37)

To a mixture of #11 (2.7 g, 9.4 mmol, 1 eq.) in dichloromethane (30 mL,0.3 M) and N,N-dimethylformamide (3 mL) were added diisopropylethylamine(3.30 mL, 18.8 mmol, 2 eq.), L-phenylalanine methyl ester hydrochloride(2.03 g, 9.40 mmol, 1.2 eq.) and HATU (4.79 g, 12.2 mmol, 1.3 eq.). Thereaction was stirred for 18 hours and then concentrated in vacuo. Theresidue was taken up in ethyl acetate (100 mL) and washed sequentiallywith 1 M hydrochloric acid (2×50 mL) and brine. The organic layer wasdried over sodium sulfate, filtered and evaporated in vacuo. The crudematerial was taken up in dichloromethane and filtered. The filtrate waspurified by silica gel chromatography (Gradient: 0% to 100% ethylacetate in heptane) to give #37 (2.76 g, 65%) as an off-white solid.LC-MS: m/z 449.3 [M+H⁺], 349.2 [(M−Boc)+H⁺] retention time=0.88 minutes;¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixture of rotamers,characteristic signals: δ 8.28 (d, J=8.2 Hz, 1H), 7.14-7.29 (m, 5H),4.50 (ddd, J=10.9, 8.1, 4.4 Hz, 1H), 3.64 (s, 3H), 3.23 (s, 3H),2.15-2.24 (m, 1H), 1.56-1.76 (m, 2H), 1.31-1.55 (m, 11H), 1.02 (d, J=6.6Hz, 3H).

Step 2 Synthesis of methylN-{(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methylpropanethioyl}-L-phenylalaninate(#38)

A mixture of #37 (1.52 g, 3.39 mmol, 1 eq.) and #21 (1.68 g, 4.41 mmol,1.3 eq.) in acetonitrile (12 mL, 0.28 M) was subjected to microwaveradiation at 100° C. for 1 hour. The mixture was partitioned betweenwater and ethyl acetate. The aqueous layer was back-extracted with ethylacetate. The combined organic layers were washed with 10% aqueous citricacid solution and with brine, dried over sodium sulfate, filtered, andconcentrated in vacuo. The material was dissolved in a small amount ofethyl acetate and concentrated onto silica in vacuo. Purification bysilica gel chromatography (Gradient: 0% to 30% ethyl acetate in heptane)provided #38 (680 mg, 43%); LC-MS: m/z 465.2 [M+H⁺], 487.3 [M+Na⁺],365.2 [(M−Boc)+H⁺], retention time=0.97 minutes; HPLC (Protocol B):465.2 [M+H⁺], 487.2 [M+Na⁺], 365.2 [(M−Boc)+H⁺], retention time=7.444minutes (purity >98%); ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ 10.23 (br d, J=7.5 Hz,1H), 7.17-7.28 (m, 5H), 5.24 (ddd, J=11, 7.5, 4.5 Hz, 1H), 3.66 (s, 3H),3.28 (s, 3H), 3.21 (dd, J=14.3, 4.4 Hz, 1H), 3.07 (dd, J=14.2, 11.2 Hz,1H), 2.65-2.74 (m, 1H), 1.54-1.71 (m, 2H), 1.37 (s, 9H), 1.17 (d, J=6.4Hz, 3H).

Step 3 Synthesis of methyl N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanethioyl}-L-phenylalaninate,hydrochloride salt (#39)

According to general procedure C, at 0° C. from #38 (660 mg, 1.42 mmol,1 eq.), dioxane (10 mL, 0.14 M) and 4 M hydrochloric acid solution indioxane (20 mL, 80 mmol, 60 eq.) was synthesized #39 (590 mg) as anoff-white solid, which was used in the next step without furtherpurification. LC-MS: m/z 365.2 [M+H⁺], retention time=0.58 minutes; ¹HNMR (400 MHz, DMSO-d₆) δ 10.67 (d, J=7.7 Hz, 1H), 9.42-9.54 (br m, 1H),8.21-8.33 (br m, 1H), 7.20-7.35 (m, 5H), 5.25 (ddd, J=11.1, 7.6, 4.4 Hz,1H), 3.76 (dd, J=8.9, 3.0 Hz, 1H), 3.68 (s, 3H), 3.39 (s, 3H), 3.24 (dd,J=14.2, 4.5 Hz, 1H), 3.13 (dd, J=14.3, 11.0 Hz, 1H), 2.93-3.09 (m, 3H),2.85-2.93 (m, 1H), 1.72-1.84 (m, 1H), 1.36-1.60 (m, 3H), 1.22 (d, J=6.6Hz, 3H).

Step 4 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N—R3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#40)

According to general procedure D, from #8 (247 mg, 0.387 mmol, 1 eq.),#39 (186 mg, ≦0.450 mmol, 1.2 eq.), dichloromethane (10 mL, 0.04 M),N,N-dimethylformamide (2 mL), HATU (176 mg, 0.464 mmol, 1.2 eq.) andtriethylamine (189 mL, 1.35 mmol, 3.5 eq.) was synthesized the crudedesired material, which was purified by silica gel chromatography(Gradient: 0% to 25% acetone in heptane) to give #40 (410 mg, 90% over 2steps) as an off-white solid. LC-MS: m/z 984.7 [M+H⁺], 1006.7 [M+Na⁺],retention time=1.15 minutes; HPLC (Protocol C): retention time=9.683minutes (purity >99%); ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ [10.19 (br d, J=7 Hz) and10.49 (br d, J=8 Hz), total 1H], 7.90 (d, J=7.5 Hz, 2H), 7.60-7.65 (m,2H), 7.38-7.45 (m, 2H), 7.29-7.35 (m, 2H), 7.14-7.28 (m, 5H), [5.20(ddd, J=11, 7, 4 Hz) and 5.35-5.43 (m), total 1H], 3.65 and 3.69 (2 s,total 3H), [1.15 (d, J=6.5 Hz) and 1.18 (d, J=6.4 Hz), total 3H].

Step 5A Synthesis ofN-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#41)

To a solution of #40 (401 mg, 0.407 mmol, 1 eq.) in tetrahydrofuran (10mL, 0.03 M) was added a solution of lithium hydroxide (24.4 mg, 1.02mmol, 2.5 eq.) in water (5 mL). After 4 hours, the reaction wasconcentrated in vacuo and then azeotroped three times with heptane. Thecrude material was dissolved in dimethyl sulfoxide (7 mL) and purifiedby reverse phase chromatography (Method C, 7 injections of 1 mL). Theappropriate fractions were concentrated (Genevac) before being dilutedwith a small amount of methanol in dichloromethane. The mixture wasconcentrated in vacuo to a glass-like solid. Diethyl ether was thenadded, followed by heptane, and the mixture was concentrated in vacuo toafford #41 (180 mg, 59%) as a white solid. LC-MS: m/z 748.6 [M+H⁺],retention time=0.68 minutes; HPLC (Protocol A): 748.4 [M+H⁺], retentiontime=6.922 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixtureof rotamers, characteristic signals: δ 12.9 and 13.1 (2 v br s, total1H), [10.12 (br d, J=8 Hz) and 10.45 (br d, J=8 Hz), total 1H],8.75-8.90 (m, 2H), 8.62-8.73 (br m, 1H), 7.13-7.29 (m, 5H), [5.20 (ddd,J=11, 7.5, 4 Hz) and 5.40 (ddd, J=11.5, 8, 4 Hz), total 1H], 4.55-4.73(m, 2H), 3.23 and 3.25 (2 s, total 3H), 3.16 and 3.18 (2 s, total 3H),2.97 and 3.01 (2 br s, total 3H), 1.13-1.20 (m, 3H), 0.73-0.81 (m, 3H).

Step 5B Synthesis ofN-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#42)

According to general procedure A, from #40 (561 mg, 0.570 mmol, 1 eq.),dichloromethane (10 mL, 0.057 M) and diethylamine (10 mL) wassynthesized #42 (348 mg, 80%) as a white solid after silica gelchromatography (Gradient: 0% to 10% methanol in dichloromethane). LC-MS:m/z 762.7 [M+H⁺], retention time=0.74 minutes; HPLC (Protocol A): 762.4[M+H⁺], retention time=7.315 minutes (purity >95%); ¹H NMR (400 MHz,DMSO-d₆), presumed to be a mixture of rotamers, characteristic signals:δ [10.20 (br d, J=7.5 Hz) and 10.50 (br d, J=8 Hz), total 1H], 7.95-8.03(m, 1H), 7.15-7.29 (m, 5H), [5.20 (ddd, J=11, 7.5, 5 Hz) and 5.39 (ddd,J=11, 7.5, 4 Hz, total 1H], [4.57 (dd, J=8.8, 8.7 Hz) and 4.61 (dd,J=8.7, 8.6 Hz), total 1H], 3.65 and 3.69 (2 s, total 3H), 3.24 and 3.25(2 s, total 3H), 3.16 and 3.17 (2 s, total 3H), 2.96 and 2.99 (2 br s,total 3H), 2.69-2.79 (m, 1H), 2.62-2.68 (m, 1H), 2.14 and 2.15 (2 br s,total 3H), [1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.5 Hz), total 3H], [0.75(t, J=7.4 Hz) and 0.76 (t, J=7.3 Hz), total 3H].

Preparation of2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,hydrochloride salt (#44) and2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,hydrochloride salt (#45)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#43)

To a solution of #32 (321 mg, 0.881 mmol, 1 eq.) in dichloromethane (5mL, 0.1 M) and N,N-dimethylformamide (1 mL) were added #39 (484 mg,≦0.769 mmol, 0.9 eq.), HATU (353 mg, 0.881 mmol, 1 eq.) anddiisopropylethylamine (463 μL, 2.64 mmol, 3 eq.). After stirring for 18hours, the mixture was diluted with dichloromethane, washed with waterand with brine, dried over magnesium sulfate, filtered, and concentratedonto silica in vacuo. The residue was purified by silica gelchromatography (Gradient: 0% to 30% acetone in heptane) to give #43 (574mg, 68% over two steps) as a white solid. LC-MS: m/z 956.6 [M+H⁺],retention time=4.49 minutes; ¹H NMR (400 MHz, CD₃OD), presumed to be amixture of rotamers, characteristic signals: δ 7.80 (d, J=7.5 Hz, 2H),7.64-7.72 (m, 2H), 7.16-7.35 (m, 7H), [5.43 (dd, J=11, 4.5 Hz) and 5.58(dd, J=11.5, 4 Hz), total 1H], 3.72 and 3.75 (2 s, total 3H), 3.34 and3.35 (2 s, total 3H), 3.26 and 3.29 (2 s, total 3H), 3.05 and 3.11 (2 brs, total 3H), 1.39 and 1.40 (2 s, total 3H), [1.24 (d, J=6.7 Hz) and1.29 (d, J=6.4 Hz), total 3H].

Step 2A Synthesis of2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,hydrochloride salt (#44)

To a solution of #43 (100 mg, 0.105 mmol, 1 eq.) in tetrahydrofuran (5mL, 0.02 M) was added a solution of lithium hydroxide (10 mg, 0.417mmol, 3 eq.) in water (3 mL). After 3 hours, the reaction wasconcentrated in vacuo and purified by reverse phase chromatography(Method C) to give a trifluoroacetic acid salt, which was dissolved inmethanol, treated with a 4 M hydrochloric acid solution in dioxane, andconcentrated in vacuo to give #44 (56 mg, 71%) as a white solid. LC-MS:m/z 720.6 [M+H⁺], retention time=0.67 minutes; HPLC (Protocol D):retention time=8.851 minutes; ¹H NMR (400 MHz, CD₃OD), presumed to be amixture of rotamers, characteristic signals: δ 7.17-7.31 (m, 5H), 3.34and 3.35 (2 s, total 3H), 3.10 and 3.16 (2 br s, total 3H), 1.62 and1.64 (2 s, total 3H), 1.53 and 1.55 (2 s, total 3H), [1.26 (d, J=6.5 Hz)and 1.30 (d, J=6.5 Hz), total 3H], 0.84-0.91 (m, 3H).

Step 2B Synthesis of2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,hydrochloride salt (#45)

According to general procedure A, from #43 (176 mg, 0.184 mmol, 1 eq.),dichloromethane (4 mL, 0.05 M) and diethylamine (4 mL) was synthesizedthe crude desired material, which was purified by reverse phasechromatography (Method C). The resulting trifluoroacetic acid salt wasdissolved in methanol, treated with a 4 M solution of hydrochloric acidin dioxane, and concentrated in vacuo to give #45 (100 mg, 70%) as awhite solid. LC-MS: m/z 734.6 [M+H⁺], retention time=0.72 minutes; ¹HNMR (400 MHz, CD₃OD), presumed to be a mixture of rotamers,characteristic signals: δ 7.18-7.31 (m, 5H), 5.41-5.47 and 5.55-5.62 (2m, total 1H), 3.73 and 3.76 (2 s, total 3H), 3.35 and 3.36 (2 s, total3H), 3.10 and 3.15 (2 br s, total 3H), 1.62 and 1.64 (2 s, total 3H),1.53 and 1.55 (2 s, total 3H), [1.25 (d, J=6.6 Hz) and 1.29 (d, J=6.5Hz), total 3H], 0.84-0.91 (m, 3H).

Preparation ofN²-[(1-Aminocyclopentyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#47)

Step 1 Synthesis ofN²-[(1-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}cyclopentyl)-carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#46)

To a solution of #19 (353 mg, 0.944 mmol, 1 eq.) in dichloromethane (10mL, 0.094 M) were added #28 (600 mg, 0.944 mmol, 0.9 eq.),diisopropylethylamine (498 μL, 2.83 mmol, 3 eq.) and HATU (444 mg, 1.13mmol, 1.2 eq.). After stirring for two days, the mixture wasconcentrated in vacuo and the residue was diluted with ethyl acetate (60mL), washed with 1 M aqueous hydrochloric acid solution and with brine,dried over sodium sulfate, filtered, and concentrated in vacuo. Theresidue was diluted with dichloromethane and filtered. The filtrate wasconcentrated under reduced pressure onto silica and purified by silicagel chromatography (Gradient: 40% to 100% ethyl acetate in heptane) togive #46 (644 mg, 69%) as a white solid. LC-MS: m/z 991.8 [M+H⁺],retention time=1.07 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ 7.89 (br d, J=7.4 Hz,2H), [7.77 (d, J=3.3 Hz) and 7.79 (d, J=3.3 Hz), total 1H], 7.66-7.76(m, 2H), [7.62 (d, J=3.3 Hz) and 7.65 (d, J=3.3 Hz), total 1H],7.37-7.44 (m, 2H), 7.11-7.36 (m, 7H), [5.38 (ddd, J=11, 8, 4 Hz) and5.48-5.57 (m), total 1H], 3.13, 3.17, 3.18 and 3.24 (4 s, total 6H),2.90 and 3.00 (2 br s, total 3H), [1.05 (d, J=6.6 Hz) and 1.09 (d, J=6.8Hz), total 3H].

Step 2 Synthesis ofN²-[(1-aminocyclopentyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#47)

To a mixture of #46 (500 mg, 0.504 mmol, 1 eq.) in tetrahydrofuran (8mL, 0.06 M) was added diethylamine (4 mL). After stirring for 18 hours,the reaction mixture was concentrated in vacuo and the residue waspurified by silica gel chromatography (Gradient: 0% to 10% methanol indichloromethane) to give #47 (374 mg, 96%) as a white solid. LC-MS: m/z769.6 [M+H⁺], retention time=0.70 minutes; ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ [8.64(br d, J=8.4 Hz) and 8.87 (br d, J=8.6 Hz), total 1H], [8.22 (br d,J=9.4 Hz) and 8.26 (br d, J=9.4 Hz), total 1H], [7.77 (d, J=3.3 Hz) and7.80 (d, J=3.3 Hz), total 1H], [7.63 (d, J=3.1 Hz) and 7.66 (d, J=3.3Hz), total 1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J=11.1, 8.5, 4.2 Hz) and5.54 (ddd, J=11.7, 8.8, 4.1 Hz), total 1H], [4.53 (dd, J=9.2, 7.6 Hz)and 4.64 (dd, J=9.2, 6.6 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s,total 6H), 2.93 and 3.03 (2 br s, total 3H), [1.05 (d, J=6.8 Hz) and1.10 (d, J=6.6 Hz), total 3H], 0.73-0.80 (m, 3H).

Preparation ofN²-[(1-Anainocyclopropyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#51) and1-anaino-N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]cyclohexanecarboxamide(#52)

Step 1 Synthesis of(2R,3R)-3-methoxy-2-methyl-N-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]-3-[(2S)-pyrrolidin-2-yl]propanamide,trifluoroacetic acid salt and(3R,4S,5S)-4-[{N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoicacid (#48)

To a solution of #16 (1.0 g, 2.11 mmol, 1 eq.) and #5 (1.22 g, 2.11mmol, 1 eq.) in dichloromethane (20 mL, 0.1 M) at 0° C. was addedtrifluoroacetic acid (6 mL). After 3 hours, the mixture was concentratedin vacuo to give the mixture #48 (1.8 g), which was used in the nextstep without further purification; LC-MS (Protocol K): m/z 374.2 [M+H⁺],retention time=2.093 minutes, 525.2 [M+H⁺], retention time=4.875minutes.

Step 2 Synthesis ofN²-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#49)

To a solution of #48 (1.8 g, ≦2.1 mmol, 1 eq.) and diethylcyanophosphonate (DEPC) (0.51 g, 3.2 mmol, 1.5 eq.) in1,2-dimethoxyethane (30 mL, 0.07 M) at 0° C. was added triethylamine(1.47 mL, 10.6 mmol, 5 eq.). After stirring at room temperature for 2hours, the mixture was concentrated in vacuo and the residue waspurified by silica gel chromatography (10% to 50% ethyl acetate inpetroleum ether) to give #49 (0.8 g, 45%). R_(f) 0.6 (10% methanol indichloromethane); LC-MS (Protocol K): m/z 881.3 [M+H⁺], 903.3 [M+Na⁺],retention time=4.837 minutes.

Step 3 Synthesis ofN-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#50)

According to general procedure A, from #49 (0.70 g, 0.79 mmol, 1 eq.),dichloromethane (15 mL, 0.05 M) and diethylamine (10 mL) was synthesized#50 (160 mg, 30%) after purification by silica gel chromatography(Gradient: 0% to 5% methanol in dichloromethane). R_(f) 0.4 (10%methanol in dichloromethane); LC-MS (Protocol K): m/z 658.3 [M+H⁺],680.3 [M+Na⁺], retention time=2.760 minutes.

Step 4A Synthesis ofN²-[(1-aminocyclopropyl)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#51)

To a solution of #50 (100 mg, 0.15 mmol, 1 eq.),bromotris(dimethylamino)phosphonium hexafluorophosphate (Brop, 70 mg,0.18 mmol, 1.2 eq.) and diisopropylethylamine (0.08 mL, 0.45 mmol, 3eq.) in dichloromethane (15 mL, 0.01 M) at 0° C. was added1-aminocyclopropanecarboxylic acid (18 mg, 0.18 mmol, 1.2 eq.). After 2hours, the mixture was quenched with water and extracted twice withethyl acetate. The combined organic layers were dried over sodiumsulfate, filtered, and concentrated in vacuo. The residue was purifiedby silica gel chromatography (Gradient: 0% to 5% methanol indichloromethane) to give #51 (45 mg, 34%). R_(f) 0.5 (10% methanol indichloromethane). LC-MS (Protocol L): m/z 741.44 [M+H⁺]; ¹H NMR (300MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ [8.64 (br d, J=8 Hz) and 8.88 (br d, J=8 Hz), total 1H],[8.16 (br d, J=9 Hz) and 8.22 (br d, J=10 Hz), total 1H], [7.77 (d,J=3.5 Hz) and 7.79 (d, J=3.5 Hz), total 1H], [7.63 (d, J=3.5 Hz) and7.65 (d, J=3 Hz), total 1H], 7.10-7.32 (m, 5H), 5.33-5.60 (m, 1H), 3.16,3.20, 3.21 and 3.26 (4 s, total 6H), 2.93 and 3.02 (2 br s, total 3H),[1.05 (d, J=6.3 Hz) and 1.10 (d, J=6.3 Hz), total 3H].

Step 4B Synthesis of1-amino-N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]cyclohexanecarboxamide (#52)

To a solution of #50 (120 mg, 0.18 mmol, 1 eq.), Brop (84 mg, 0.21 mmol,1.2 eq.) and diisopropylethylamine (0.1 mL, 0.54 mmol, 3 eq.) indichloromethane (15 mL, 0.009 M) at 0° C. was added1-aminocyclohexanecarboxylic acid (31 mg, 0.21 mmol, 1.2 eq.). After 2hours, the mixture was quenched with water and extracted twice withethyl acetate. The combined organic layers were dried over sodiumsulfate, filtered, and concentrated in vacuo. The residue was purifiedby silica gel chromatography (Gradient: 0% to 5% methanol indichloromethane) to give #52 (50 mg, 35%). R_(f) 0.6 (10% methanol indichloromethane). LC-MS (Protocol K): m/z 783.79 [M+H⁺]; ¹H NMR (400MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ [8.64 (br d, J=8 Hz) and 8.87 (br d, J=9 Hz), total 1H],8.18-8.28 (m, 1H), [7.77 (d, J=3.5 Hz) and 7.80 (d, J=3.3 Hz), total1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.3 Hz), total 1H], 7.12-7.31(m, 5H), 5.35-5.43 and 5.49-5.57 (2 m, total 1H), [4.51 (dd, J=9, 8 Hz),and 4.61 (dd, J=9, 7 Hz), total 1H], 3.16, 3.19, 3.21 and 3.25 (4 s,total 6H), 2.93 and 3.02 (2 br s, total 3H), [1.05 (d, J=6.8 Hz) and1.10 (d, J=6.8 Hz), total 3H].

Preparation of2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#54)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#53)

According to general procedure D, from #32 (2.05 g, 2.83 mmol, 1 eq.) indichloromethane (20 mL, 0.1 M) and N,N-dimethylformamide (3 mL), theamine #19 (2.5 g, 3.4 mmol, 1.2 eq.), HATU (1.29 g, 3.38 mmol, 1.2 eq.)and triethylamine (1.57 mL, 11.3 mmol, 4 eq.) was synthesized the crudedesired material, which was purified by silica gel chromatography(Gradient: 0% to 55% acetone in heptane), producing #53 (2.42 g, 74%) asa solid. LC-MS: m/z 965.7 [M+H⁺], 987.6 [M+Na⁺], retention time=1.04minutes; HPLC (Protocol A): m/z 965.4 [M+H⁺], retention time=11.344minutes (purity >97%); ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ 7.86-7.91 (m, 2H), [7.77(d, J=3.3 Hz) and 7.79 (d, J=3.2 Hz), total 1H], 7.67-7.74 (m, 2H),[7.63 (d, J=3.2 Hz) and 7.65 (d, J=3.2 Hz), total 1H], 7.38-7.44 (m,2H), 7.30-7.36 (m, 2H), 7.11-7.30 (m, 5H), [5.39 (ddd, J=11.4, 8.4, 4.1Hz) and 5.52 (ddd, J=11.7, 8.8, 4.2 Hz), total 1H], [4.49 (dd, J=8.6,7.6 Hz) and 4.59 (dd, J=8.6, 6.8 Hz), total 1H], 3.13, 3.17, 3.18 and3.24 (4 s, total 6H), 2.90 and 3.00 (2 br s, total 3H), 1.31 and 1.36 (2br s, total 6H), [1.05 (d, J=6.7 Hz) and 1.09 (d, J=6.7 Hz), total 3H].

Step 2 Synthesis of2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#54)

According to general procedure A, from #53 (701 mg, 0.726 mmol) indichloromethane (10 mL, 0.07 M) was synthesized the crude desiredmaterial, which was purified by silica gel chromatography (Gradient: 0%to 10% methanol in dichloromethane). The residue was diluted withdiethyl ether and heptane and was concentrated in vacuo to afford #54(406 mg, 75%) as a white solid. LC-MS: m/z 743.6 [M+H⁺], retentiontime=0.70 minutes; HPLC (Protocol A): m/z 743.4 [M+H⁺], retentiontime=6.903 minutes, (purity >97%); ¹H NMR (400 MHz, DMSO-d₆), presumedto be a mixture of rotamers, characteristic signals: δ [8.64 (br d,J=8.5 Hz) and 8.86 (br d, J=8.7 Hz), total 1H], [8.04 (br d, J=9.3 Hz)and 8.08 (br d, J=9.3 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d,J=3.2 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.2 Hz), total1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J=11, 8.5, 4 Hz) and 5.53 (ddd,J=12, 9, 4 Hz), total 1H], [4.49 (dd, J=9, 8 Hz) and 4.60 (dd, J=9, 7Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.93 and 3.02(2 br s, total 3H), 1.21 (s, 3H), 1.13 and 1.13 (2 s, total 3H), [1.05(d, J=6.7 Hz) and 1.10 (d, J=6.7 Hz), total 3H], 0.73-0.80 (m, 3H).

Preparation of2-Methylalanyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide,acetic acid salt (#56)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#55)

To a solution of #24 (104 mg, 0.256 mmol, 1 eq.) in dichloromethane (10mL, 0.094 M) were added #32 (156 mg, 0.256 mmol, 0.9 eq.),diisopropylethylamine (135 μL, 0.768 mmol, 3 eq.) and HATU (120 mg,0.307 mmol, 1.2 eq.). After stirring for 18 hours, the mixture wasconcentrated in vacuo and the residue was diluted with ethyl acetate (10mL), washed with 1 M aqueous hydrochloric acid solution (2×5 mL) andwith brine, dried over sodium sulfate, filtered, and concentrated invacuo. The residue was diluted with dichloromethane and filtered. Thefiltrate was concentrated under reduced pressure onto silica andpurified by silica gel chromatography (Gradient: 0% to 100% ethylacetate in heptane) to give #55 (44 mg, 19%) as a white solid. LC-MS:m/z 884.5 [M+2H⁺], retention time=1.04 minutes.

Step 2 Synthesis of2-methylalanyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide,acetic acid salt (#56)

To a mixture of #55 (44 mg, 0.050 mmol, 1 eq.) in tetrahydrofuran (1 mL,0.05 M) was added diethylamine (0.5 mL). After stirring for 18 hours,the reaction mixture was concentrated in vacuo and the residue waspurified by reverse phase chromatography (Method B) to give #56 (16.2mg, 49%) as a solid. LC-MS: m/z 660.8 [M+H⁺], retention time=2.23minutes; HPLC (Protocol A): m/z 660.5 [M+H⁺], 682.4

[M+Na⁺], retention time=6.865 minutes.

Preparation of2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1-phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#60)

Step 1 Synthesis of 1-(1-phenylcyclopropyl)methanamine #@1

To a solution of 1-phenylcyclopropanecarbonitrile (50 g, 0.34 mol, 1eq.) in tetrahydrofuran (500 mL, 0.7 M) at 0° C. was added lithiumaluminum hydride (23 g, 0.35 mol, 1.03 eq.). The reaction mixture wasstirred at 0° C. for one hour and then at reflux for one hour. Thereaction mixture was then cooled down and quenched with water (23 mL)and a 15% aqueous sodium hydroxide solution (69 mL). The mixture wasfiltered and concentrated in vacuo to afford #*1 (36 g, 72%). LC-MS: m/z148.1 [M+H⁺], retention time=0.86 minutes; ¹H NMR (400 MHz, CDCl₃) δ7.2-7.4 (m, 5H), 2.78 (s, 2H), 1.19 (br s, 2H), 0.72-0.84 (m, 4H).

Step 2 Synthesis of tert-butyl(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1-phenylcyclopropyl)methyl]amino}propyl]pyrrolidine-1-carboxylate(#57)

According to general procedure D, from #11 (2.15 g, 7.48 mmol, 1.1 eq.)in dichloromethane (20 mL, 0.3 M) and N,N-dimethylformamide (4 mL),1-(1-phenylcyclopropyl)methanamine #@1 (1.001 g, 6.799 mmol, 1 eq.),HATU (3.10 g, 8.16 mmol, 1.2 eq.) and triethylamine (2.84 mL, 20.4 mmol,3 eq.) was synthesized the crude desired material, which was purified bysilica gel chromatography (Gradient: 0% to 100% ethyl acetate inheptane), producing #57 (1.93 g, 68%) as a solid. HPLC (Protocol A at45° C.): m/z 417.3 [M+H⁺], retention time=10.575 minutes; ¹H NMR (400MHz, DMSO-d₆), presumed to be a mixture of rotamers: δ 7.75-7.81 (m,1H), 7.20-7.27 (m, 4H), 7.12-7.19 (m, 1H), 3.33-3.62 and 3.71-3.80 (brmultiplets, total 4H), 3.28 (s, 3H), 2.97-3.17 (br m, 2H), 2.14-2.24 (m,1H), 1.67-1.80 (br m, 2H), 1.45-1.65 (m, 2H), 1.41 (s, 9H), 1.00 (d,J=6.6 Hz, 3H), 0.67-0.93 (m, 4H).

Step 3 Synthesis of(2R,3R)-3-methoxy-2-methyl-N-[(1-phenylcyclopropyl)methyl]-3-[(2S)-pyrrolidin-2-yl]propanamide,hydrochloride salt (#58)

According to general procedure C, from #57 (566 mg, 1.36 mmol, 1 eq.) indioxane (4 mL, 0.3 M) and 4 M hydrochloric acid solution in dioxane (4mL, 16 mmol, 11.7 eq.) was synthesized #58 (466 mg, 97%); LC-MS: m/z318.2 [M+H⁺], 339.2 [M+Na⁺], retention time=0.56 minute; ¹H NMR (400MHz, DMSO-d₆) δ 9.53 (br s, 1H), 8.48 (br s, 1H), 8.11 (br dd, J=5.7,5.6 Hz, 1H), 7.23-7.30 (m, 4H), 7.14-7.21 (m, 1H), 3.58 (dd, J=7.5, 3.9Hz, 1H), 3.50 (dd, J=13.7, 6.3 Hz, 1H), 3.34 (s, 3H), 3.21-3.29 (br m,1H), 3.18 (dd, J=13.8, 5.0 Hz, 1H), 3.04-3.13 (br m, 2H), 2.42-2.50 (m,1H), 1.56-1.89 (m, 4H), 1.04 (d, J=6.9 Hz, 3H), 0.71-0.91 (m, 4H).

Step 4 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1-phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#59)

According to general procedure D, from #32 (550 mg, 0.902 mmol, 1 eq.),#58 (350 mg, 0.992 mmol, 1.1 eq.) dichloromethane (10 mL, 0.08 M) andN,N-dimethylformamide (2 mL), HATU (446 mg, 1.17 mmol, 1.3 eq.) andtriethylamine (0.503 mL, 3.61 mmol, 4 eq.) was synthesized the crudedesired material, which was purified by silica gel chromatography(Gradient: 0% to 30% acetone in heptane), producing #59 (618 mg, 69%) asan off-white solid. LC-MS: m/z 908.7 [M+H⁺], 930.7 [M+Na⁺], retentiontime=1.07 minutes; HPLC (Protocol B at 45° C.): m/z 908.5 [M+H⁺],retention time=8.721 minutes (purity >97%); ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ 7.89 (d,J=7.5 Hz, 2H), 7.38-7.44 (m, 2H), 7.30-7.36 (m, 2H), [4.49 (dd, J=8.5,7.8 Hz) and 4.59 (dd, J=8.7, 6.9 Hz), total 1H], 4.18-4.26 (m, 3H),3.93-4.01 (br m, 1H), 3.23 and 3.26 (2 s, total 3H), 3.16 and 3.16 (2 s,total 3H), 2.91 and 3.05 (2 br s, total 3H), 1.36 and 1.37 (2 br s,total 3H), 1.30 and 1.32 (2 br s, total 3H), [1.00 (d, J=6.7 Hz) and1.02 (d, J=6.6 Hz), total 3H], 0.67-0.78 (m, 7H).

Step 5 Synthesis of2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1-phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#60)

According to general procedure A, from #59 (605 mg, 0.666 mmol, 1 eq.)dichloromethane (10 mL, 0.067 M) and diethylamine (10 mL) wassynthesized #60 (379 mg, 83%); HPLC (Protocol A at 45° C.) m/z 685.5[M+H⁺], retention time=7.072 minutes; ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ [8.03(br d, J=9.6 Hz) and 8.07 (br d, J=9.4 Hz), total 1H], [7.74 (br dd,J=7, 4 Hz) and 7.99 (br dd, J=5.9, 5.7 Hz), total 1H], 7.20-7.27 (m,4H), 7.11-7.17 (m, 1H), [4.49 (dd, J=9, 7 Hz) and 4.58 (dd, J=9, 7.5Hz), total 1H], 3.96-4.04 (br m, 1H), 3.24 and 3.27 (2 s, total 3H),3.18 and 3.19 (2 s, total 3H), 2.93 and 3.07 (2 br s, total 3H), 1.20and 1.21 (2 s, total 3H), 1.12 and 1.14 (2 s, total 3H), [1.00 (d, J=6.7Hz) and 1.03 (d, J=6.7 Hz), total 3H].

Preparation of2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#66)

Step 1 Synthesis of cyclohepta-2,4,6-trien-1-ylacetonitrile (#61)

To a solution of anhydrous acetonitrile (3.12 mL, 56.2 mmol, 1 eq.) intetrahydrofuran (281 mL, 0.2 M) was added lithium diisopropylamine (1.8M in heptane/ethylbenzene/tetrahydrofuran, 31.2 mL, 56.2 mmol, 1 eq.) at−78° C. After 20 minutes at −78° C., tropylium tetrafluoroborate (10 g,56 mmol, 1 eq.) was added. After 10 minutes, the reaction wasconcentrated in vacuo and the residue was diluted with ethyl acetate andwashed with water. The organic layer was dried over sodium sulfate,filtered, and concentrated in vacuo to provide a brown oil, which waspurified by silica gel chromatography (Gradient: 0% to 10% ethyl acetatein heptane) to provide #61 (1.88 g, 25%) as a yellow oil. ¹H NMR (400MHz, CDCl₃) δ 6.69-6.71 (m, 2H), 6.27-6.32 (m, 2H), 5.28-5.33 (m, 2H),2.61 (d, J=7.2 Hz, 2H), 2.26-2.34 (m, 1H).

Step 2 Synthesis of 2-(cyclohepta-2,4,6-trien-1-yl)ethanamine (#62)

To a suspension of lithium aluminum hydride (911 mg, 24.0 mmol, 1.4 eq.)in anhydrous diethyl ether (75 mL, 0.23 M) at 0° C. was slowly added,drop-wise over 15 minutes, a solution of #61 (2.25 g, 17.2 mmol, 1 eq.)in diethyl ether (15 mL). The reaction was warmed to room temperature.After 5 hours, the reaction was cooled to 0° C. and quenched by additionof water (1 mL), then filtered through a small pad of Celite and washedwith methanol. The filtrate was dried over sodium sulfate, filtered, andconcentrated in vacuo to provide #62 (1.683 g, 73%) as a golden oil.LC-MS: m/z 136.1 [M+H⁺], retention time=0.23 minutes; ¹H NMR (400 MHz,CDCl₃) δ 6.64-6.67 (m, 2H), 6.16-6.21 (m, 2H), 5.16-5.21 (m, 2H),2.84-2.89 (m, 2H), 1.86-1.92 (m, 2H), 1.62-1.70 (m, 1H).

Step 3 Synthesis of tert-butyl(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate(#63)

To a solution of #11 (3.57 g, 12.4 mmol, 1 eq.) in dichloromethane (100mL, 0.1 M) and N,N-dimethylformamide (4 mL) was added HATU (5.36 g, 13.7mmol, 1.1 eq.). After 20 minutes, triethylamine (5.20 mL, 37.3 mmol, 3eq.) was added, followed by #62 (1.68 g, 12.4 mmol, 1 eq.), and themixture was stirred for 18 hours. The reaction was concentrated in vacuoand the residue was taken up in ethyl acetate and washed with water (50mL). The aqueous layer was back-extracted with ethyl acetate (3 times)and the combined organic layers were dried, filtered, and concentratedin vacuo to provide a brown oil, which was purified by silica gelchromatography (Gradient: 0% to 100% ethyl acetate in heptane) toprovide #63 (2.95 g, 59% yield) as a viscous oil. LC-MS: m/z 405.4[M+H⁺], 427.4 [M+Na⁺], retention time=0.75 minutes; ¹H NMR (400 MHz,CDCl₃), presumed to be a mixture of rotamers: δ 6.63-6.68 (m, 2H),6.16-6.23 (m, 2H), 5.19 (br dd, J=9.0, 5.8 Hz, 2H), 3.51-3.63 and3.71-3.90 (2 br multiplets, total 3H), 3.42 (s, 3H), 3.18-3.29 and3.34-3.47 (2 br multiplets, total 3H), 2.27-2.45 (br m, 1H), 1.6-2.00(m, 7H), 1.47 and 1.50 (2 br s, total 9H), 1.16-1.29 (br m, 3H).

Step 4 Synthesis of(2R,3R)—N-[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanamide,hydrochloride salt (#64)

Intermediate #63 (400 mg, 0.989 mmol, 1 eq.) was treated with a 4 Msolution of hydrochloric acid in dioxane (10 mL, 40 mmol, 40 eq.). After1 hour, the reaction mixture was concentrated in vacuo and the residuewas taken up in dichloromethane and washed with 1 M sodium hydroxidesolution. The aqueous layer was back-extracted with dichloromethane andthe combined organic layers were dried over sodium sulfate, filtered,and concentrated in vacuo to provide #64 (301 mg, quantitative) as abrown oil, which slowly began to solidify upon standing. LC-MS: m/z305.3 [M+H⁺], retention time=0.54 minutes; HPLC (Protocol G): retentiontime=4.848 minutes; ¹H NMR (400 MHz, CDCl₃), characteristic signals: δ6.64-6.67 (m, 2H), 6.16-6.22 (m, 2H), 6.08-6.14 (br m, 1H), 5.16-5.22(m, 2H), 3.44 (s, 3H), 3.31 (dd, J=6.3, 4.5 Hz, 1H), 2.98-3.04 (m, 1H),2.94 (ddd, J=10.5, 7.2, 5.6 Hz, 1H), 2.81 (ddd, J=10.5, 7.7, 6.7 Hz,1H), 2.57 (qd, J=7.1, 4.5 Hz, 1H), 1.90-1.97 (m, 2H), 1.49-1.55 (m, 1H),1.18 (d, J=7.1 Hz, 3H).

Step 5 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#65)

According to general procedure D, from #32 (678 mg, 0.937 mmol, 1 eq.)in dichloromethane (9.37 mL, 0.1 M), the amine #64 (300 mg, 0.985 mmol,1.1 eq.), HATU (427 mg, 1.12 mmol, 1.2 eq.) and diisopropylethylamine(494 μL, 2.81 mmol, 3 eq.) was synthesized the crude desired material,which was purified by silica gel chromatography (Gradient: 0% to 50%acetone in heptane), producing #65 (546 mg, 65%) as a solid. LC-MS: m/z896.7 [M+H⁺], 918.7 [M+Na⁺], retention time=1.06 minutes.

Step 6 Synthesis of2-methylalanyl-N-[(3R,4S,55)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#66)

To a solution of #65 (540 mg, 0.603 mmol, 1 eq.) in dichloromethane (10mL, 0.06 M) was added triethylamine (10 mL) and the reaction mixture wasstirred for 2 hours. The mixture was concentrated in vacuo and theresidue was purified by silica gel chromatography (Gradient: 0% to 10%methanol in dichloromethane) to give #66 (347 mg, 85%) as a colorlesssolid. HPLC (Protocol A at 45° C.): m/z 674.5 [M+H⁺], retentiontime=7.015 minutes. ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixtureof rotamers, characteristic signals: δ [8.03 (br d, J=9 Hz) and 8.05 (brd, J=9 Hz), total 1H], [7.77 (br dd, J=5.5, 5.5 Hz) and 7.98 (br dd,J=5.5, 5.5 Hz), total 1H], 6.54-6.65 (m, 2H), 6.10-6.19 (m, 2H),5.11-5.19 (m, 2H), [4.48 (dd, J=9, 8 Hz) and 4.54 (dd, J=9, 7.5 Hz),total 1H], 3.94-4.04 (br m, 1H), 3.26 and 3.29 (2 s, total 3H), 3.17 and3.19 (2 s, total 3H), 2.93 and 3.06 (2 br s, total 3H), 1.20 and 1.21 (2s, total 3H), 1.12 and 1.13 (2 s, total 3H), [1.04 (d, J=6.8 Hz) and1.07 (d, J=6.7 Hz), total 3H].

Preparation of2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#69) and2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#70)

Step 1 Synthesis of methylN-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenylalaninate,hydrochloride salt (#67)

According to general procedure C, from #37 (2.39 g, 5.33 mmol, 1 eq.),dioxane (10 mL, 0.53 M) and a 4 M hydrochloric acid solution in dioxane(10 mL, 40 mmol, 7.5 eq.) was synthesized #67 (2.21 g) as a white solid,which was used in the next step without further purification. LC-MS: m/z349.2 [M+H⁺], retention time=0.53 minutes; ¹H NMR (400 MHz, DMSO-d₆) δ9.45-9.58 (br m, 1H), 8.63 (d, J=8.1 Hz, 1H), 8.51-8.62 (br m, 1H),7.25-7.33 (m, 4H), 7.18-7.25 (m, 1H), 4.50 (ddd, J=10.8, 8.1, 4.5 Hz,1H), 3.65 (s, 3H), 3.54 (dd, J=6.8, 4.5 Hz, 1H), 3.20 (s, 3H), 3.11 (dd,J=13.8, 4.5 Hz, 1H), 2.99-3.14 (br m, 3H), 2.89 (dd, J=13.8, 10.9 Hz,1H), 2.44-2.50 (m, 1H, assumed; partially obscured by solvent peak),1.77-1.89 (m, 1H), 1.60-1.73 (m, 2H), 1.46-1.57 (m, 1H), 1.05 (d, J=6.8Hz, 3H).

Step 2 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#68)

According to general procedure D, from #32 (353 mg, 0.488 mmol, 1 eq.)in dichloromethane (10 mL, 0.04 M), amine #67 (271 mg, ≦0.588 mmol, 1.3eq.), HATU (223 mg, 0.586 mmol, 1.2 eq.) and diisopropylethylamine (238μL, 1.71 mmol, 3.5 eq.) was synthesized the crude desired material,which was purified by silica gel chromatography (Gradient: 0% to 40%acetone in heptane), affording #68 (404 mg, 88% over two steps) as asolid. LC-MS: m/z 940.7 [M+H⁺], 962.7 [M+Na⁺], retention time=1.04minutes; HPLC (Protocol C): retention time=9.022 minutes; ¹H NMR (400MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ [8.25 (br d, J=8 Hz) and 8.48 (br d, J=8 Hz), total 1H], 7.89(d, J=7.4 Hz, 2H), 7.67-7.75 (m, 2H), 7.38-7.44 (m, 2H), 7.31-7.36 (m,2H), 7.14-7.24 (m, 5H), 4.43-4.69 (m, 3H), 4.17-4.26 (m, 3H), 3.91-3.99(br m, 1H), 3.63 and 3.65 (2 s, total 3H), 3.19 and 3.24 (2 s, total3H), 3.14 and 3.15 (2 s, total 3H), 2.90 and 2.99 (2 br s, total 3H),1.36 and 1.37 (2 br s, total 3H), 1.30 and 1.32 (2 s, total 3H), [1.02(d, J=6.8 Hz) and 1.06 (d, J=6.6 Hz), total 3H].

Step 3A Synthesis of2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#69)

To a solution of #68 (143 mg, 0.152 mmol, 1 eq.) in tetrahydrofuran (5mL, 0.02 M) was added a solution of lithium hydroxide (9.10 mg, 0.378mmol, 2.5 eq.) in water (3 mL). After 5 hours, the reaction wasconcentrated in vacuo, azeotroped three times with heptane, dissolved indimethyl sulfoxide (2.2 mL) and purified by reverse phase chromatography(Method C) to give #69 (56 mg, 52%). HPLC (Protocol A at 45° C.): 704.4[M+H⁺], retention time=6.623 minutes; ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ8.08-8.22 and 8.37-8.49 (2 m, total 5H), 7.12-7.28 (m, 5H), 3.18, 3.20and 3.24 (3 s, total 6H), 2.95 and 3.04 (2 br s, total 3H), 1.52 and1.53 (2 s, total 3H), 1.39 and 1.41 (2 s, total 3H), [1.02 (d, J=6.8 Hz)and 1.05 (d, J=6.6 Hz), total 3H], 0.74-0.81 (m, 3H).

Step 3B Synthesis of2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#70)

According to general procedure A, from #68 (240 mg, 0.255 mmol, 1 eq.),dichloromethane (10 mL, 0.026 M) and diethylamine (10 mL) wassynthesized #70 (120 mg, 65%) as a white solid/glass mix after silicagel chromatography (Gradient: 0% to 10% methanol in dichloromethane).HPLC (Protocol A at 45° C.): m/z 762.7 [M+H⁺], 740.4 [M+Na⁺], retentiontime=6.903 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixtureof rotamers, characteristic signals: δ [8.26 (d, J=8.1 Hz) and 8.49 (d,J=8.3 Hz), total 1H], [8.03 (d, J=9.5 Hz) and 8.07 (d, J=9.5 Hz), total1H], 7.14-7.27 (m, 5H), 3.63 and 3.67 (2 s, total 3H), 3.16, 3.18, 3.20and 3.25 (4 s, total 6H), 2.92 and 3.01 (2 br s, total 3H), 1.20 and1.22 (2 s, total 3H), 1.12 and 1.13 (2 s, total 3H), [1.02 (d, J=6.8 Hz)and 1.06 (d, J=6.7 Hz), total 3H], 0.74-0.80 (m, 3H).

Preparation ofN²-[(3-Aminooxetan-3-yl)carbonyl]-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide,acetic acid salt (#75)

Step 1 Synthesis of 3-[(tert-butoxycarbonyl)amino]oxetane-3-carboxylicacid (#71)

To 1-aminooxetane-3-carboxylic acid (1.00 g, 8.54 mmol, 1 eq.) indioxane (15 mL, 0.5 M) was added a solution of sodium hydroxide (1.55 g,38.7 mmol, 4.5 eq.) in water (15 mL) followed by di-tert-butyldicarbonate (2.09 g, 9.29 mmol, 1.1 eq.) A white solid formed. Thereaction was stirred for 18 hours and then concentrated in vacuo. Theresidue was taken up in ethyl acetate and washed with 1 M aqueoushydrochloric acid solution and with brine. The organic layer was driedover sodium sulfate, filtered, and concentrated in vacuo to give #71(633 mg, 38%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆), presumed tobe a mixture of rotamers: δ 12.93 (br s, 1H), 7.59 and 7.93 (2 br s,total 1H), 4.71-4.78 (m, 2H), 4.47 (d, J=6.4 Hz, 2H), 1.30 and 1.38 (2s, total 9H).

Step 2 Synthesis ofN²-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#72)

To #@5 (9.47 g, 18.0 mmol, 1 eq.) and #24 (5.90 g, 18.0 mmol, 1 eq.) indichloromethane (250 mL, 0.072 M) were added diisopropylethylamine (9.52mL, 54.2 mmol, 3 eq.) and HATU (8.49 g, 21.7 mmol, 1.2 eq.). Thereaction was stirred for 18 hours and then concentrated in vacuo. Theresidue was taken up in ethyl acetate (300 mL) and was washed with 1 Maqueous hydrochloric acid solution (2×100 mL) and with brine. Theorganic layer was dried over sodium sulfate, filtered, and concentratedin vacuo. The residue was taken up in dichloromethane (250 mL) andfiltered. The filtrate was concentrated in vacuo onto silica andpurified by silica gel chromatography (Gradient: 0% to 50% acetone inheptane) to provide #72 (11.61 g, 81%) as a light yellow solid. LC-MS:m/z 797.6 [M+H⁺], 819.6 [M+Na⁺], retention time=1.06 minutes; ¹H NMR(400 MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ 3.26 and 3.28 (2 s, total 3H), 3.18 and 3.20 (2 s, total 3H),2.95 and 3.10 (2 br s, total 3H), 1.01-1.09 (m, 3H), 0.67-0.78 (m, 3H).

Step 3 Synthesis ofN-{(3R,4S,5S)-3-methoxy-1—R2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#73)

To #72 (5.16 g, 6.47 mmol, 1 eq.) in tetrahydrofuran (10 mL, 0.65 M) wasadded diethylamine (10 mL). After 2 hours, the reaction was concentratedin vacuo and the residue was purified by silica gel chromatography(Gradient: 0% to 10% methanol in dichloromethane) to give #73 (2414 mg,65%). LC-MS: m/z 576.5 [M+H⁺], retention time=0.64 minutes; ¹H NMR (400MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ 7.80-7.88 and 7.99-8.10 (2 m, total 1H), 7.14-7.31 (m, 5H),3.17 and 3.18 (2 s, total 3H), 2.87 and 3.03 (2 br s, total 3H),1.02-1.08 (m, 3H).

Step 4 Synthesis ofN²-({3-[(tert-butoxycarbonyl)amino]oxetan-3-yl}carbonyl)-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#74)

To #73 (100 mg, 0.174 mmol, 1 eq.) in dichloromethane (4 mL, 0.04) andN,N-dimethylformamide (0.5 mL) was added #71 (45.2 mg, 0.208 mmol, 1.2eq.), followed by diisopropylethylamine (92 μL, 0.521 mmol, 3 eq.) andHATU (102 mg, 0.260 mmol, 1.5 eq.). After 16 hours, the reaction wasconcentrated in vacuo and the residue was taken up in ethyl acetate (6mL) and washed with 1 M aqueous hydrochloric acid solution (2×2 mL) andwith brine. The organic layer was dried over sodium sulfate, filtered,and concentrated in vacuo. The crude material was purified by reversephase chromatography (Method C) to give #74 (140 mg), which was used inthe next step without further purification. LC-MS: m/z 774.7 [M+H⁺],796.6 [M+Na⁺], retention time=0.91 minute.

Step 5 Synthesis ofN²-[(3-aminooxetan-3-yl)carbonyl]-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide,acetic acid salt (#75)

To #74 (140 mg, ≦0.181 mmol, 1 eq.) in dichloromethane (3 mL, 0.06 M)was added trifluoroacetic acid (1 mL). After 1 hour, the reaction wasconcentrated in vacuo and the residue was taken up in ethyl acetate (6mL) and washed with saturated aqueous sodium bicarbonate solution (2 mL)and with brine. The organic layer was dried over sodium sulfate,filtered, and concentrated in vacuo. Half of the crude material waspurified by reverse phase chromatography (Method B) to give #75 (16 mg,26%, over two steps). LC-MS: m/z 674.6 [M+H⁺], retention time=0.68minutes; HPLC (Protocol A at 45° C.): m/z 674.5 [M+H⁺], retentiontime=7.128 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixtureof rotamers, characteristic signals: δ 7.80-7.87 and 8.02-8.07 (2 m,total 2H), 7.23-7.30 (m, 2H), 7.14-7.22 (m, 3H), 4.28-4.33 (m, 2H),3.96-4.04 (br m, 1H), 3.17 and 3.19 (2 s, total 3H), 2.96 and 3.10 (2 brs, total 3H), [1.04 (d, J=7.0 Hz) and 1.07 (d, J=6.6 Hz), total 3H].

Preparation ofN,2-Dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#79) andN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#80)

Step 1 Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanethioyl}-L-phenylalaninate(#76)

According to general procedure D, from #@5 (260 mg, 0.648 mmol, 1 eq.),#39 (340 mg, ≦0.629 mmol, 1 eq.), dichloromethane (10 mL, 0.065 M), HATU(296 mg, 0.778 mmol, 1.2 eq.) and diisopropylethylamine (339 μL, 1.94mmol, 3 eq.) was synthesized the crude desired material, which waspurified by silica gel chromatography (Gradient: 0% to 40% acetone inheptane) to give #76 (466 mg, 83% over two steps) as a solid. LC-MS: m/z871.5 [M+H⁺], 893.5 [M+Na⁺], retention time=1.10 minutes; HPLC (ProtocolC): retention time=9.249 minutes (purity >99%); ¹H NMR (400 MHz,DMSO-d₆), presumed to be a mixture of rotamers, characteristic signals:δ [10.19 (br d, J=7.4 Hz) and 10.49 (br d, J=7.8 Hz), total 1H], 7.89(br d, J=7.4 Hz, 2H), 7.68-7.75 (m, 2H), 7.54-7.60 (m, 1H), 7.41 (br dd,J=7.4, 7.4 Hz, 2H), 7.28-7.36 (m, 2H), 7.15-7.28 (m, 5H), [5.20 (ddd,J=10.9, 7.3, 4.4 Hz) and 5.34-5.43 (m), total 1H], 3.65 and 3.69 (2 s,total 3H), 3.24 and 3.25 (2 s, total 3H), 3.17 (br s, 3H), 2.93 and 2.98(2 br s, total 3H), [1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.6 Hz), total3H].

Step 2 Synthesis of methylN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanethioyl}-L-phenylalaninate(#77)

According to general procedure A, from #76 (460 mg, 0.528 mmol, 1 eq.)tetrahydrofuran (8 mL, 0.07 M) and diethylamine (8 mL) was synthesized#77 (399 mg), which was used in the next step without furtherpurification; LC-MS m/z 649.5 [M+H⁺], retention time=0.73 minutes; ¹HNMR (400 MHz, DMSO-d₆), presumed to be a mixture of rotamers,characteristic product signals: δ [10.20 (d, J=7.4 Hz) and 10.49 (d,J=7.4 Hz), total 1H], 7.15-7.28 (m, 5H), [5.20 (ddd, J=10.9, 7.2, 4.5Hz) and 5.34-5.42 (m), total 1H], 3.65 and 3.68 (2 s, total 3H), 3.24and 3.25 (2 s, total 3H), 3.15 and 3.15 (2 s, total 3H), 2.83 and 2.88(2 br s, total 3H), [1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.6 Hz), total3H].

Step 3 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#78)

According to general procedure D, from #77 (399 mg, ≦0.52 mmol, 1 eq.),N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanine (213 mg, 0.628mmol, 1.2 eq.), dichloromethane (5 mL, 0.1 M), HATU (239 mg, 0.628 mmol,1.2 eq.) and diisopropylethylamine (282 μL, 1.62 mmol, 3.1 eq.) wassynthesized the crude desired material, which was purified by silica gelchromatography (Gradient: 0% to 50% acetone in heptane), providing #78(231 mg, 46% over two steps). LC-MS: m/z 970.7 [M+H⁺], 992.6 [M+Na⁺],retention time=1.11 minutes; HPLC (Protocol C): retention time=9.260minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixture ofrotamers, characteristic signals: δ [10.19 (d, J=7.4 Hz) and 10.47 (d,J=7.8 Hz), total 1H], 7.89 (d, J=7.4 Hz, 2H), 7.61-7.67 (m, 2H), 7.41(br dd, J=7.4, 7.4 Hz, 2H), 7.14-7.36 (m, 8H), [5.20 (ddd, J=11, 7, 5Hz) and 5.38 (ddd, J=11, 8, 4 Hz), total 1H], [4.41 (dd, J=8.6, 8.4 Hz)and 4.46 (dd, J=8.2, 8.2 Hz), total 1H], 3.65 and 3.68 (2 s, total 3H),3.23 and 3.24 (2 s, total 3H), 3.13 (br s, 3H), 2.88 and 2.93 (2 br s,total 3H), 2.84 and 2.85 (2 s, total 3H), 1.31 and 1.32 (2 s, total 3H),[1.15 (d, J=6.6 Hz) and 1.18 (d, J=6.4 Hz), total 3H].

Step 4A Synthesis ofN,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#79)

According to general procedure A, from #78 (223 mg, 0.230 mmol, 1 eq.),dichloromethane (6 mL, 0.04 M) and diethylamine (6 mL) was synthesized#79 (146 mg, 85%) as a white solid after silica gel chromatography(Gradient: 0% to 5% methanol in heptane then 0% to 10% methanol indichloromethane). HPLC (Protocol A at 45° C.): 749.4 [M+H⁺], retentiontime=7.315 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixtureof rotamers, characteristic signals: δ [10.20 (d, J=7.6 Hz) and 10.50(d, J=8.0 Hz), total 1H], 7.79-7.88 (m, 1H), 7.15-7.29 (m, 5H), [5.20(ddd, J=11, 7, 4 Hz) and 5.38 (ddd, J=11, 8, 4 Hz), total 1H], [4.50(dd, J=8.8, 8.6 Hz) and 4.56 (dd, J=9, 8 Hz), total 1H], 3.65 and 3.69(2 s, total 3H), 3.24 and 3.25 (2 s, total 3H), 3.16 (br s, 3H), 2.93and 2.97 (2 br s, total 3H), 2.10 and 2.11 (2 s, total 3H).

Step 4B Synthesis ofN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#80)

To a solution of #78 (170 mg, 0.175 mmol, 1 eq.) in tetrahydrofuran (3mL, 0.04 M) was added a solution of lithium hydroxide (12.6 mg, 0.525mmol, 3 eq.) in water (1.5 mL). After stirring overnight, the solventwas removed in vacuo. The residue was azeotroped three times withheptane. The residue was then diluted with dimethyl sulfoxide (2.2 mL)and purified by reverse phase chromatography (Method C) to afford #80(74 mg, 58%) as a solid. LC-MS: m/z 734.6 [M+H⁺], retention time=0.69minutes; HPLC (Protocol A at 45° C.): 734.4 [M+H⁺], retention time=6.903minutes (purity >96%); ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ 12.9 and 13.1 (2 v br s,total 1H), [10.12 (d, J=7.4 Hz) and 10.46 (d, J=7.8 Hz), total 1H],8.77-8.89 (br m, 2H), [8.47 (d, J=8.6 Hz) and 8.51 (d, J=8.6 Hz), total1H], 7.21-7.29 (m, 4H), 7.14-7.21 (m, 1H), [5.16-5.23 (m) and 5.38 (ddd,J=11.3, 8.2, 3.9 Hz), total 1H], [4.51 (dd, J=9.0, 9.0 Hz) and 4.57 (dd,J=9.4, 8.6 Hz), total 1H], 3.24 and 3.24 (2 s, total 3H), 3.18 and 3.19(2 s, total 3H), 2.96 and 3.00 (2 br s, total 3H), 1.51 and 1.53 (2 s,total 3H), 1.40 and 1.42 (2 s, total 3H), 1.14-1.19 (m, 3H), 0.74-0.81(m, 3H).

Preparation ofN,2-Dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#84)

Step 1 Synthesis ofN²-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#81)

According to general procedure D, from #@5 (620 mg, 1.18 mmol, 1 eq.)dichloromethane (10 mL, 0.1 M), amine #18 (604 mg, 1.42 mmol, 1.2 eq.),diisopropylethylamine (618 μL, 3.54 mmol, 3 eq.) and HATU (539 mg, 1.42mmol, 1.2 eq.) was synthesized the crude desired material, which waspurified by silica gel chromatography (Gradient: 0% to 30% acetone inheptane) to give #81 (737 mg, 58%). HPLC (Protocol C): retentiontime=9.235 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixtureof rotamers, characteristic signals: δ [10.54 (br d, J=8 Hz) and 10.81(br d, J=8 Hz), total 1H], 7.89 (d, J=7.6 Hz, 2H), [7.80 (d, J=3.3 Hz)and 7.83 (d, J=3.1 Hz), total 1H], 7.70-7.75 (m, 2H), [7.64 (d, J=3.1Hz) and 7.68 (d, J=3.3 Hz), total 1H], 7.55-7.60 (m, 1H), 7.38-7.44 (m,2H), 7.13-7.35 (m, 7H), [6.31 (ddd, J=11, 8, 4.5 Hz) and 6.40-6.48 (m),total 1H], 3.23 and 3.24 (2 s, total 3H), 3.17 and 3.22 (2 s, total 3H),2.94 and 3.01 (2 br s, total 3H), [1.14 (d, J=6.4 Hz) and 1.17 (d, J=6.2Hz), total 3H].

Step 2 Synthesis ofN-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#82)

According to general procedure A, from #81 (733 mg, 0.818 mmol, 1 eq.)in dichloromethane (7 mL, 0.1 M) and diethylamine (7 mL) was synthesized#82 (670 mg), which was used in the next step without furtherpurification. LC-MS: m/z 674.5 [M+H⁺], retention time=1.29 minutes; ¹HNMR (400 MHz, DMSO-d₆), presumed to be a mixture of rotamers,characteristic product signals: δ [10.55 (br d, J=8 Hz) and 10.84 (br d,J=8 Hz), total 1H], [7.64 (d, J=3.1 Hz) and 7.69 (d, J=3.3 Hz), total1H], 7.13-7.33 (m, 5H), 6.27-6.35 and 6.38-6.47 (2 m, total 1H), 3.23and 3.25 (2 s, total 3H), 3.15 and 3.19 (2 s, total 3H), 2.84 and 2.91(2 br s, total 3H).

Step 3 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#83).

According to general procedure D, from #82 (670 mg, ≦0.818 mmol, 1 eq.),dichloromethane (5 mL, 0.16 M),N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanine (304 mg, 0.896mmol, 1.1 eq.), HATU (372 mg, 0.978 mmol, 1.2 eq.) anddiisopropylethylamine (440 μL, 2.53 mmol, 3.1 eq.) was synthesized thecrude desired material, which was purified by silica gel chromatography(Gradient: 0% to 30% acetone in heptane) to give #83 (556 mg, 69% overtwo steps). LC-MS: m/z 994.7 [M+H⁺], retention time=0.69 minutes; HPLC(Protocol C): retention time=9.333 minutes (purity >98%); ¹H NMR (400MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: δ [10.53 (br d, J=8 Hz) and 10.80 (br d, J=8 Hz), total 1H],7.86-7.91 (m, 2H), [7.80 (d, J=3.3 Hz) and 7.82 (d, J=3.2 Hz), total1H], [7.64 (d, J=3.2 Hz) and 7.68 (d, J=3.2 Hz), total 1H], 7.62-7.66(m, 2H), 7.38-7.44 (m, 2H), 7.28-7.36 (m, 5H), 7.19-7.26 (m, 2H),7.12-7.17 (m, 1H), [6.31 (ddd, J=11, 8, 4.5 Hz) and 6.44 (ddd, J=11,8.5, 4.5 Hz), total 1H], [4.42 (dd, J=9, 8 Hz) and 4.48 (dd, J=8, 8 Hz),total 1H], 3.22 and 3.24 (2 s, total 3H), 3.13 and 3.17 (2 s, total 3H),2.89 and 2.97 (2 br s, total 3H), 2.84 and 2.85 (2 s, total 3H), [1.13(d, J=6.4 Hz) and 1.16 (d, J=6.4 Hz), total 3H].

Step 4 Synthesis ofN,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#84)

According to general procedure A, from #83 (552 mg, 0.555 mmol, 1 eq.)in dichloromethane (10 mL, 0.05 M) and diethylamine (10 mL) wassynthesized the crude desired material, which was diluted with methanol,concentrated in vacuo onto silica, and purified by silica gelchromatography (Gradient: 0% to 10% methanol in dichloromethane) to give#84 (406 mg, 95%) as a white solid. LC-MS: m/z 772.8 [M+H⁺], retentiontime=1.35 minutes; HPLC (Protocol A): 774.4 [M+H⁺], retention time=7.390minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixture ofrotamers, characteristic signals: δ [10.54 (br d, J=8 Hz) and 10.81 (brd, J=8 Hz), total 1H], 7.78-7.84 (m, 2H), [7.65 (d, J=3.1 Hz) and 7.69(d, J=3.3 Hz), total 1H], 7.29-7.34 (m, 2H), 7.20-7.28 (m, 2H),7.14-7.19 (m, 1H), 6.27-6.35 and 6.40-6.48 (2 m, total 1H), [4.51 (dd,J=9, 8 Hz) and 4.57 (dd, J=9, 8 Hz), total 1H], 3.24 and 3.25 (2 s,total 3H), 3.16 and 3.21 (2 s, total 3H), 2.94 and 3.00 (2 br s, total3H), 2.09 and 2.10 (2 s, total 3H), 1.08 and 1.09 (2 s, total 3H),0.73-0.80 (m, 3H).

N,2-Dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#88)

Step 1 Synthesis ofN²-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#85)

To a mixture of #@5 (5.48 g, 10.4 mmol, 1 eq.) and #19 (3.90 g, 10.4mmol, 1 eq.) in dichloromethane (50 mL, 0.2M) was addeddiisopropylethylamine (5.51 mL, 31.3 mmol, 3 eq.) followed by HATU (4.91g, 12.5 mmol, 1.2 eq.). After stirring overnight, the reaction mixturewas concentrated in vacuo. The residue was taken up in ethyl acetate(100 mL) and washed with 1 M aqueous hydrochloric acid solution (2×30mL) and with brine (30 mL). The organic layer was dried over sodiumsulfate, filtered, and concentrated in vacuo. The residue was taken upin dichloromethane and filtered; the filtrate was purified by silica gelchromatography (Gradient; 0% to 50% acetone in heptane) to afford #85(7.20 g, 78%) as a solid. LC-MS: m/z 880.6 [M+H⁺], retention time=1.07minutes.

Step 2 Synthesis ofN-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#86)

According to general procedure A, from #85 (5.00 g, 5.68 mmol, 1 eq.) intetrahydrofuran (10 mL, 0.56 M) and diethylamine (3 mL) was synthesizedthe crude desired material, which was purified by silica gelchromatography (Gradient: 0% to 10% methanol in dichloromethane) to give#86 (2.952 g, 79%) as a solid. LC-MS: m/z 658.5 [M+H⁺], 680.5 [M+Na⁺]retention time=0.66 minute; ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ [8.64 (br d, J=8.4 Hz)and 8.90 (br d, J=8.8 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d,J=3.3 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.3 Hz), total1H], 7.12-7.31 (m, 5H), [5.39 (ddd, J=11.2, 8.4, 4.2 Hz) and 5.54 (ddd,J=11.9, 8.9, 4.0 Hz), total 1H], 3.15, 3.19, 3.20 and 3.26 (4 s, total6H), 2.86 and 2.98 (2 br s, total 3H), [1.06 (d, J=6.6 Hz) and 1.11 (d,J=6.6 Hz), total 3H].

Step 3 Synthesis ofN-(tert-butoxycarbonyl)-N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#87)

To a mixture of #86 (80.3 mg, 0.122 mmol, 1 eq.) in dichloromethane (4mL, 0.03 M) was added N-(tert-butoxycarbonyl)-N,2-dimethylalanine (29.1mg, 0.134 mmol, 1.1 eq.) followed by diisopropylethylamine (64 μL, 0.365mmol, 3 eq.) and HATU (71.7 mg, 0.183 mmol, 1.5 eq.) After stirringovernight, the reaction mixture was concentrated in vacuo. The residuewas taken up in ethyl acetate (6 mL) and washed with 1 M aqueoushydrochloric acid solution (2×2 mL) and with brine. The organic solventwas dried over sodium sulfate, filtered, and concentrated in vacuo. Theresidue was taken up in dichloromethane and filtered; the filtrate wasconcentrated in vacuo onto silica and purified by silica gelchromatography (Gradient: 0% to 50% acetone in heptane) to afford #87(58 mg, 50%) as a white solid. LC-MS: m/z 857.7 [M+H⁺], 879.7 [M+Na⁺],retention time=0.99 minute; ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ [8.64 (br d, J=8 Hz) and8.87 (br d, J=9 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d, J=3.3Hz), total 1H], [7.63 (d, J=3.2 Hz) and 7.66 (d, J=3.2 Hz), total 1H],7.13-7.31 (m, 5H), [6.95 (br d, J=8 Hz) and 7.06 (br d, J=8 Hz), total1H], 5.35-5.42 and 5.51-5.58 (2 m, total 1H), 3.15, 3.19, 3.20 and 3.26(4 s, total 6H), 2.94 and 3.03 (2 br s, total 3H), 2.83 and 2.84 (2 s,total 3H), [1.05 (d, J=6.7 Hz) and 1.11 (d, J=6.7 Hz), total 3H].

Step 4 Synthesis ofN,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#88)

To a mixture of #87 (58 mg, 0.068 mmol, 1 eq.) in dichloromethane (8 mL)was added trifluoroacetic acid (2 mL). After stirring overnight, thereaction mixture was concentrated in vacuo. The residue was taken up inethyl acetate (10 mL), washed with saturated aqueous sodium bicarbonatesolution, dried over sodium sulfate, filtered, and concentrated in vacuoto give #88 (52 mg, quantitative). LC-MS 757.6 [M+H⁺], retentiontime=0.69 minute; ¹H NMR (400 MHz, DMSO-d₆), presumed to be a mixture ofrotamers, characteristic signals: δ [8.64 (br d, J=8.6 Hz) and 8.87 (brd, J=8.6 Hz), total 1H], 7.80-7.85 (m, 1H), [7.77 (d, J=3.3 Hz) and 7.80(d, J=3.1 Hz), total 1H], [7.63 (d, J=3.1 Hz) and 7.66 (d, J=3.3 Hz),total 1H], 7.20-7.31 (m, 4H), 7.13-7.19 (m, 1H), [5.39 (ddd, J=11, 8.5,4 Hz) and 5.49-5.56 (m), total 1H], [4.51 (dd, J=9, 8 Hz) and 4.61 (dd,J=9, 8 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.94and 3.03 (2 br s, total 3H), 2.10 and 2.10 (2 s, total 3H), 1.16 (br s,3H), 1.04-1.12 (m, 6H), 0.72-0.80 (m, 3H).

Preparation ofN²-(3-Amino-2,2-dimethylpropanoyl)-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#95)

Step 1 Synthesis of3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-2,2-dimethylpropanoic acid(#93)

To 3-amino-2,2-dimethylpropanoic acid, hydrochloride salt (250 mg, 1.63mmol, 1 eq.) in dichloromethane (4 mL, 0.4 M) was addeddiisopropylethylamine (859 μL, 4.88 mmol, 3 eq.) followed by(9H-fluoren-9-ylmethoxy)carbonyl chloride (473 mg, 1.79 mmol, 1.1 eq.)The reaction was stirred for 18 hours and then concentrated in vacuo.The residue was taken up in ethyl acetate (3 mL) and washed with 1 Maqueous hydrochloric acid solution (2×1 mL) and with brine. The organiclayer was dried over sodium sulfate, filtered, and purified by silicagel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) togive #93 (250 mg, 45%) as an oil. ¹H NMR (400 MHz, DMSO-d₆) δ 12.22 (s,1H), 7.89 (d, J=7.4 Hz, 2H), 7.72 (d, J=7.4 Hz, 2H), 7.38-7.44 (m, 2H),7.27-7.35 (m, 3H), 4.18-4.30 (m, 3H), 3.16 (d, J=6.2 Hz, 2H), 1.05 (s,6H).

Step 2 Synthesis ofN²-(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-2,2-dimethylpropanoyl)-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#94)

To #86 (100 mg, 0.152 mmol, 1 eq.) in dichloromethane (4 mL, 0.038 M)and N,N-dimethylformamide (0.5 mL) was added #93 (51.6 mg, 0.152 mmol, 1eq.) followed by diisopropylethylamine (80.0 μL, 0.457 mmol, 3 eq.) andHATU (89.8 mg, 0.229 mmol, 1.5 eq.). The reaction was stirred for 18hours and then concentrated in vacuo. The residue was taken up in ethylacetate (6 mL) and was washed with 1 M aqueous hydrochloric acidsolution (2×2 mL) and with brine. The organic layer was dried oversodium sulfate, filtered, and concentrated in vacuo. The residue wastaken up in dichloromethane (250 mL) and filtered; the filtrate wasconcentrated in vacuo onto silica and purified by silica gelchromatography (Gradient: 0% to 50% acetone in heptane) to provide #94(90 mg, 60%) as a white solid. LC-MS: m/z 979.8 [M+H⁺], 1002.7 [M+Na⁺],retention time=1.15 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic product signals: δ [8.64 (br d,J=8.6 Hz) and 8.86 (br d, J=8.6 Hz), total 1H], 7.86-7.91 (m, 2H), [7.77(d, J=3.3 Hz) and 7.79 (d, J=3.3 Hz), total 1H], 7.67-7.73 (m, 2H),[7.63 (d, J=3.3 Hz) and 7.65 (d, J=3.3 Hz), total 1H], 6.87-6.95 (m,1H), [5.39 (ddd, J=11, 8, 4 Hz) and 5.52 (ddd, J=11.5, 9, 4 Hz), total1H], [4.44 (dd, J=8.4, 8.4 Hz) and 4.55 (dd, J=8.4, 8.4 Hz), total 1H],3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.96 and 3.06 (2 br s, total3H), 0.69-0.77 (m, 3H).

Step 3 Synthesis ofN²-(3-amino-2,2-dimethylpropanoyl)-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#95)

To #94 (86 mg, 0.088 mmol, 1 eq.) in tetrahydrofuran (2 mL, 0.04 M) wasadded diethylamine (10 mL). After stirring overnight, the reaction wasconcentrated in vacuo and the residue was purified by reverse phasechromatography (Method C) to give #95 (55 mg, 72%). LC-MS: m/z 757.5[M+H⁺], retention time=0.74 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumedto be a mixture of rotamers, characteristic signals: δ [8.66 (br d, J=8Hz) and 8.92 (br d, J=9 Hz), total 1H], [7.91 (br d, J=8 Hz) and 7.97(br d, J=9 Hz), total 1H], [7.78 (d, J=3.3 Hz) and 7.81 (d, J=3.1 Hz),total 1H], 7.65-7.74 (br m, 3H), [7.63 (d, J=3.3 Hz) and 7.67 (d, J=3.3Hz), total 1H], 7.12-7.31 (m, 5H), [5.35-5.42 (m) and 5.45-5.52 (m),total 1H], [4.44 (dd, J=9, 9 Hz) and 4.55 (dd, J=9, 9 Hz), total 1H],3.17, 3.20, 3.22 and 3.25 (4 s, total 6H), 2.96 and 3.05 (2 br s, total3H), 1.25 and 1.25 (2 s, total 3H), 1.14 and 1.15 (2 s, total 3H), [1.06(d, J=6.6 Hz) and 1.10 (d, J=6.4 Hz), total 3H], 0.72-0.80 (m, 3H).

Preparation ofN²-(3-Amino-2,2-dimethylpropanoyl)-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#97)

Step 1 Synthesis ofN²-(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-2,2-dimethylpropanoyl)-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#96)

To #73 (100 mg, 0.174 mmol, 1 eq.) in dichloromethane (4 mL, 0.04 M) andN,N-dimethylformamide (0.5 mL) was added #93 (59.1 mg, 0.174 mmol, 1eq.), followed by diisopropylethylamine (92 μL, 0.52 mmol, 3 eq.) andHATU (102 mg, 0.260 mmol, 1.5 eq.). The reaction was stirred for 18hours and then concentrated in vacuo. The residue was taken up in ethylacetate (6 mL) and was washed with 1 M aqueous hydrochloric acidsolution (2×2 mL) and with brine. The organic layer was dried oversodium sulfate, filtered, and concentrated in vacuo. Purification bysilica gel chromatography (Gradient: 0% to 50% acetone in heptane)provided #96 (102 mg, 65%) as a white solid. LC-MS: m/z 896.7 [M+H⁺],918.8 [M+Na⁺], retention time=1.14 minutes; ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic product signals: δ7.88 (d, J=7.4 Hz, 2H), [7.83 (br dd, J=6, 5 Hz) and 8.03 (br dd, J=6, 5Hz), total 1H], 7.67-7.73 (m, 2H), 7.36-7.48 (m, 3H), 7.22-7.35 (m, 4H),7.13-7.21 (m, 3H), 6.86-6.96 (m, 1H), [4.44 (dd, J=8.6, 8.6 Hz) and 4.50(dd, J=8.6, 8.6 Hz), total 1H], 3.18, 3.19, 3.26 and 3.29 (4 s, total6H), 2.96 and 3.11 (2 br s, total 3H), 0.70-0.77 (m, 3H).

Step 2 Synthesis ofN²-(3-amino-2,2-dimethylpropanoyl)-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#97)

To #96 (98 mg, 0.11 mmol, 1 eq.) in tetrahydrofuran (2 mL, 0.04 M) wasadded diethylamine (0.5 mL). After stirring overnight, the reaction wasconcentrated in vacuo and the residue was purified by reverse phasechromatography (Method C) to give #97 (58 mg, 68%). LC-MS: m/z 674.4[M+H⁺], 696.4 [M+Na⁺], retention time=0.74 minutes; HPLC (Protocol A):674.5 [M+H⁺], retention time=7.072 minutes; ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ [7.92(br d, J=8 Hz) and 7.97 (br d, J=8 Hz), total 1H], [7.86 (br dd, J=6, 5Hz) and 8.07 (br dd, J=6, 5 Hz), total 1H], 7.64-7.74 (br m, 3H),7.15-7.29 (m, 5H), [4.44 (dd, J=9, 9 Hz) and 4.50 (dd, J=9, 9 Hz), total1H], 3.26 and 3.29 (2 s, total 3H), 3.18 and 3.20 (2 s, total 3H), 2.96and 3.10 (2 br s, total 3H), 1.24 and 1.25 (2 s, total 3H), 1.14 and1.16 (2 s, total 3H), 1.02-1.07 (m, 3H), 0.73-0.80 (m, 3H).

Preparation of2-methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino]propyl}pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-vaLinamide,trifluoroacetic acid salt (#98)

To a mixture 1-(tert-butoxycarbonyl)-2-methyl-L-proline (65.1 mg, 0.284mmol, 1.1 eq.) and #86 (170 mg, 0.258 mmol, 1 eq.) in dichloromethane (5mL, 0.03 M) was added HATU (0.108 mg, 0.284 mmol, 1.1 eq.) followed bydiisopropylethylamine (139 μL, 0.800 mmol, 3.1 eq.). After stirringovernight, the reaction mixture was cooled to 0° C., dichloromethane (3mL) was added followed by the slow addition of trifluoroacetic acid (2mL). The reaction mixture was stirred at 0° C. for 5 minutes, allowed towarm to room temperature and then stirred at room temperature for 30minutes before being concentrated in vacuo. The residue was azeotropedtwo times with heptane, diluted with a small amount of dichloromethaneand methanol before being concentrated in vacuo onto silica The residuewas purified by silica gel chromatography (Gradient: 0% to 10% methanolin dichloromethane) and then by reverse phase chromatography (Method C)to afford #98 (128 mg, 56%) as a white solid. LC-MS: m/z 769.4 [M+H⁺],retention time=1.28 minutes; HPLC (Protocol A at 45° C.) m/z 769.4[M+H⁺], retention time=7.146 minutes (purity >98%); ¹HNMR (400 MHz,DMSO-d₆), presumed to be a mixture of rotamers, characteristic signals:δ 9.03-9.15 (br m, 1H), 8.77-8.86 (br m, 1H), 8.69-8.76 (m, 1H), [8.66(d, J=8.2 Hz) and 8.92 (d, J=8.6 Hz), total 1H], [7.78 (d, J=3.1 Hz) and7.80 (d, J=3.5 Hz), total 1H], [7.63 (d, J=3.1 Hz) and 7.67 (d, J=3.1Hz), total 1H], 7.12-7.31 (m, 5H), [5.38 (ddd, J=11, 8, 4 Hz) and 5.47(ddd, J=11, 9, 4 Hz), total 1H], [4.46 (dd, J=9.4, 9.0 Hz) and 4.55 (dd,J=9.0, 8.6 Hz), total 1H], 3.17, 3.20, 3.22 and 3.25 (4 s, total 6H),2.98 and 3.04 (2 br s, total 3H), [1.06 (d, J=7.0 Hz) and 1.09 (d, J=6.6Hz), total 3H], 0.73-0.80 (m, 3H).

Preparation of methyl amino(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)acetate,hydrochloride salt (#102)

Step 1 Synthesis of ethyl(acetylamino)(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)cyanoacetate (#99)

Sodium (464 mg, 20.2 mmol, 1.2 eq.) was allowed to react with absoluteethanol (40 mL, 0.42 M); to the resulting mixture was added ethyl2-(acetylamino)-2-cyanoacetate (3.44 g, 20.2 mmol, 1.2 eq.). After 20minutes at 60° C., 7-bromobicyclo[4.2.0]octa-1,3,5-triene (3.092 g,16.89 mmol, 1 eq.) was added and the reaction mixture was heated atreflux overnight, then filtered and concentrated in vacuo. The residuewas diluted with water and extracted with ethyl acetate. The organiclayer was washed with brine, dried over sodium sulfate, filtered, andconcentrated in vacuo to give a dark oil, which was purified by silicagel chromatography (Gradient: 0% to 50% ethyl acetate in heptane) togive #99 (4.38 g) as a yellow gum. LC-MS: m/z 273.2 [M+H⁺], retentiontime=2.36 minutes.

Step 2 Synthesis of(acetylamino)(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)acetic acid (#100)

To a mixture of #99 (4.38 mg, <16.1 mmol, 1 eq.) in methanol (30 mL,0.53 M) was added a 1 N aqueous solution of sodium hydroxide (38 mL, 38mmol, 2.4 eq.). The reaction mixture was heated at reflux overnight,then concentrated in vacuo, diluted with water (40 mL), and acidifiedwith a 1 N aqueous solution of hydrochloric acid (40 mL). The aqueouslayer was extracted with dichloromethane (3×30 mL). The combined organiclayers were dried over sodium sulfate, filtered and concentrated invacuo. The resulting oil was purified by silica gel chromatography(Solvent A: dichloromethane; Solvent B: 20% methanol in dichloromethanecontaining 0.02% trifluoroacetic acid; Gradient: 0% to 40% B) then bysupercritical fluid chromatography (Column: Chiralpak AD-H, 250×21 mm;Eluent: 85:15 carbon dioxide/methanol; Flow Rate: 65 g/min; Detection:210 nm; Instrument: Berger minigram preparative SFC system). The secondeluting peak was isolated to give #100 (600 mg, 17% over two steps) as asingle enantiomer (retention time=3.37 minutes, purity >99%). LC-MS: m/z220.3 [M+H⁺], retention time=2.10 minutes; ¹H NMR (400 MHz, CD₃OD) δ7.14-7.24 (m, 2H), 7.03-7.09 (m, 2H), 4.59 (d, J=8.6 Hz, 1H), 3.87 (ddd,J=8.5, 5.3, 2.4 Hz, 1H), 3.35 (dd, J=14.5, 5.4 Hz, 1H, assumed;partially obscured by solvent peak), 3.10 (dd, J=14.4, 2.4 Hz, 1H), 2.00(s, 3H). Optical rotation: [α]_(D) ²⁵+70.9° (c 0.67, methanol)

Step 3 Synthesis of amino(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)aceticacid, hydrochloride salt (#101)

A mixture of #100 (200 mg, 0.912 mmol, 1 eq.) and 6 N aqueoushydrochloric acid (12.3 mL, 73.8 mmol, 81 eq.) was heated at refluxovernight. The reaction mixture was concentrated in vacuo to give thesingle enantiomer #101 (195 mg) as an off-yellow solid, which was usedin the next step without further purification.

Step 4 Synthesis of methylamino(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)acetate, hydrochloride salt(#102)

To a mixture of #101 (195 mg, <0.913 mmol, 1 eq.) in methanol (20 mL,0.04 M) was added thionyl chloride (0.666 mL, 9.13 mmol, 10 eq.). Aftertwo hours at reflux, the reaction mixture was concentrated in vacuo togive the single enantiomer #102 (175 mg, 84% over two steps) as alight-colored solid. LC-MS: m/z 192.3 [M+H⁺], retention time=0.80minutes; GC-MS: m/z 192 [M+H⁺], retention time=3.206 minutes; ¹H NMR(400 MHz, CD₃OD) δ 7.24-7.33 (m, 2H), 7.11-7.18 (m, 2H), 4.40 (d, J=6.9Hz, 1H), 3.99-4.05 (m, 1H), 3.78 (s, 3H), 3.46 (dd, J=14.8, 5.4 Hz, 1H),3.23 (dd, J=14.8, 2.5 Hz, 1H).

Preparation of(2R,3R)-3-Methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yflpropanoic acid,hydrochloride salt (#103)

To a mixture of #11 (4.09 g, 14.2 mmol, 1 eq.) in cyclopentyl methylether (10 mL, 0.14 M) was added a 4 N solution of hydrogen chloride indioxane (37 mL, 100 mmol, 7 eq.). After three hours, the reactionmixture was concentrated in vacuo and azeotroped three times withheptane to give #103 (1000 mg, 31%) as a gum, which was used in the nextstep without further purification. ¹H NMR (400 MHz, DMSO-d₆) δ9.92-10.06 (br s, 1H), 8.66-8.80 (br s, 1H), 3.89 (dd, J=5.2, 4.9 Hz,1H), 3.43-3.53 (m, 1H), 3.39 (s, 3H), 3.06-3.17 (m, 2H), 2.66 (qd,J=7.1, 4.6 Hz, 1H), 1.71-2.03 (m, 4H), 1.11 (d, J=7.1 Hz, 3H).

Preparation of2-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[1-(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-A-N-methyl-L-valinamide,trifluoroacetic acid salt (#107) and2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[bicyclo[4.2.0]octa-1,3,5-trien-7-yhcarboxy)methyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-A-N-methyl-L-valinamide,trifluoroacetic acid salt (#108)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-5-methyl-1-oxo-1-(pentafluorophenoxy)heptan-4-yl]-N-methyl-L-valinamide(#104)

To #32 (4.00 g, 6.56 mmol, 1 eq.) in dichloromethane (20 mL, 0.33 M) andpyridine (1.06 mL, 13.1 mmol, 2 eq.) was added drop-wisepentafluorophenyl trifluoroacetate (2.25 mL, 13.1 mmol, 2 eq.). Thereaction mixture was stirred for one hour.

To a second flask containing #32 (360 mg, 0.59 mmol) in dichloromethane(0.6 mL, 1 M) and pyridine (0.095 mL, 1.2 mmol, 2 eq.) was addeddrop-wise pentafluorophenyl trifluoroacetate (0.203 mL, 1.18 mmol). Thisreaction mixture was stirred for 15 minutes.

The two reaction mixtures were combined, washed twice with 1 N aqueoushydrochloric acid, dried over sodium sulfate, filtered and concentratedin vacuo. The resulting yellow oil was dissolved in ethyl acetate,pre-adsorbed onto silica gel and purified by silica gel chromatography(Gradient: 0% to 40% ethyl acetate in heptane) to give #104 (4.6 g, 83%)as a white foam containing some impurities. LC-MS: m/z 798.3 [M+Na⁺],retention time=1.23 minutes.

Step 2 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#105)

To a mixture of #104 (2.00 g, <2.58 mmol, 1 eq.) in dichloromethane (6mL, 0.4 M) was added a solution of #103 (483 mg, 2.16 mmol, 1 eq.) indichloromethane (2 mL) followed by diisopropylethylamine (1.35 mL, 7.73mmol, 3 eq.). The reaction mixture was stirred for 16 hours, thenadsorbed onto silica and purified by silica gel chromatography(Gradient: 0% to 20% methanol in dichloromethane) to give #105 (1.67 g,83%) as a white foam. Fractions containing the desired product withimpurities (0.571 g) were collected separately.

The above reaction and purification were repeated in a similar fashionusing #104 (2.60 g, <3.35 mmol, 1 eq.), #103 (750 mg, 3.35 mmol, 1 eq.),dichloromethane (10 mL, 0.3 M) and diisopropylethylamine (1.35 mL, 7.73mmol, 2.3 eq.) to give #105 (2.4 g, 92%) as a tan foam. Fractionscontaining impure product (1.7 g) were combined with the previous impurefractions and purified as described above to afford additional #105(1.30 g, quantitative yield for both reactions over two steps). LC-MS:m/z 779.3 [M+H⁺], 802.3 [M+Na⁺], retention time=1.05 minutes.

Step 3 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[1-(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#106)

To a mixture of #105 (225 mg, 0.289 mmol, 1 eq.) in dichloromethane (15mL, 0.02 M) and N,N-dimethylformamide (1 mL) was added HATU (136 mg,0.347 mmol, 1.2 eq.). After five minutes, a solution of amine #102 (72.4mg, 0.318 mmol, 1 eq.) and diisopropylamine (203 mL, 1.16 mmol, 3 eq.)in dichloromethane (5 mL) was added. After 24 hours, the reactionmixture was washed with brine, dried over sodium sulfate, filtered,concentrated in vacuo onto silica gel, and purified by silica gelchromatography (Gradient: 0% to 50% acetone in heptane) to give thesingle enantiomer #106 (210 mg, 76%) as a clear oil. LC-MS: m/z 953.1[M+H⁺], retention time=3.99 minutes; ¹H NMR (400 MHz, CDCl₃), presumedto be a mixture of rotamers, characteristic signals: 7.76 (d, J=7.5 Hz,2H), 7.57-7.64 (m, 2H), 7.40 (dd, J=7.5, 7.4 Hz, 2H), 7.28-7.34 (m, 2H),4.82-4.88 (m, 1H), 3.95-4.01 (m, 1H), 3.76 and 3.82 (2 s, total 3H),3.30, 3.31, 3.34 and 3.35 (4 s, total 6H), [1.20 (d, J=7.0 Hz) and 1.20(d, J=7.0 Hz), total 3H].

Step 4A Synthesis of2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[1-(bicyclo[4.2.0]octa-1,3,5-trien-7-yl)-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#107)

According to general procedure A, from #106 (25 mg, 0.026 mmol, 1 eq.)in dichloromethane (10 mL, 0.003 M) and diethylamine (4 mL) wassynthesized the crude desired material, which was purified by reversephase chromatography (Method C) to give the single enantiomer #107 (16mg, 73%) as a solid. LC-MS: m/z 730.8 [M+H⁺], retention time=2.13minutes; HPLC (Protocol N): retention time=9.889 minutes.

Step 4B Synthesis of2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[bicyclo[4.2.0]octa-1,3,5-trien-7-yl(carboxy)methyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#108)

The single enantiomer #108 (94.5 mg, 57%) was synthesized from #106 (190mg, 0.200 mmol) according to a procedure similar to the one describedfor synthesis of #41 from #40. LC-MS: m/z 716.8 [M+H⁺], retentiontime=2.06 minutes; HPLC (Protocol N): retention time=9.137 minutes.

Preparation of2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#112)

Step 1 Synthesis of tert-butyl(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate(#109)

To a solution of #11 (2.00 g, 6.96 mmol, 1 eq.) in dichloromethane (21mL, 0.3 M) and N,N-dimethylformamide (3 mL) was added HATU (3270 mg,8.35 mmol, 1.2 eq.). After two minutes, the amine(1R,2S)-(+)-norephedrine (1.07 mg, 6.96 mmol, 1 eq.) and triethylamine(1.94 mL, 13.9 mmol, 2 eq.) were added. After two hours, the reactionmixture was diluted with ethyl acetate (100 mL), washed with a 1 Maqueous solution of hydrochloric acid and with brine, dried over sodiumsulfate, filtered, concentrated in vacuo, and purified by silica gelchromatography (Gradient: 0% to 60% ethyl acetate in heptane) to provide#109 (2.18 g, 74%) as a white solid. LC-MS: m/z 321.3 [(M−Boc)+H⁺],retention time=3.14 minutes; ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ 7.64 (d, J=8.6 Hz, 1H),7.24-7.33 (m, 4H), 7.15-7.21 (m, 1H), 5.35 (br d, J=5 Hz, 1H), 4.45 (brdd, J=5, 5 Hz, 1H), 3.91-4.00 (m, 1H), 3.30-3.39 (m, 1H), 3.26 (s, 3H),2.94-3.07 (m, 1H), 2.04-2.14 (m, 1H), 1.46-1.78 (m, 4H), 1.40 (s, 9H),0.97-1.04 (m, 6H).

Step 2 Synthesis of(2R,3R)—N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanamide,trifluoroacetic acid salt (#110)

According to general procedure C, at 0° C. from #109 (414 mg, 0.984mmol, 1 eq.), dioxane (5 mL, 0.2 M) and a 4 M solution of hydrogenchloride in dioxane (15 mL, 60 mmol, 60 eq.) was synthesized the crudedesired compound, which was purified by reverse phase chromatography(Method C) to give #110 (120 mg, 34%) as a viscous liquid. LC-MS: m/z321.1 [M+H⁺], retention time=0.55 minutes; ¹H NMR (400 MHz, DMSO-d₆),characteristic signals: δ 7.90 (d, J=8.6 Hz, 1H), 7.28-7.36 (m, 4H),7.20-7.27 (m, 1H), 4.46 (d, J=6.2 Hz, 1H), 3.48 (dd, J=8.6, 2.3 Hz, 1H),3.38 (s, 3H), 2.92-3.16 (m, 3H), 2.24-2.35 (m, 1H), 1.49-1.88 (m, 4H),1.09 (d, J=6.6 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H).

Step 3 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N—[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#111)

According to general procedure D, from #32 (140 mg, 0.230 mmol, 1 eq.),#110 (110 mg, 0.253 mmol, 1.1 eq.), dichloromethane (3 mL, 0.08 M),N,N-dimethylformamide (0.5 mL), HATU (96.2 mg, 0.253 mmol, 1.1 eq) andtriethylamine (96 μL, 0.69 mmol, 3 eq.) was synthesized the crudedesired product, which was purified by silica gel chromatography(Gradient: 0% to 40% acetone in heptane) to give #111 (220 mg, 95%).LC-MS: m/z 912.4 [M+H⁺], 935.4 [M+Na⁺], retention time=2.15 minutes;HPLC (Protocol B): m/z 912.5 [M+H⁺], 934.5 [M+Na⁺], retentiontime=10.138 minutes (purity >94%); ¹H NMR (400 MHz, DMSO-d₆), presumedto be a mixture of rotamers, characteristic signals: δ 7.89 (d, J=7.8Hz, 2H), 7.66-7.75 (m, 2H), 7.41 (dd, J=7.4, 7.4 Hz, 2H), 7.12-7.20 (m,1H), [5.33 (d, J=4.7 Hz) and 5.38 (d, J=4.7 Hz), total 1H], 3.15, 3.18,3.22 and 3.23 (4 s, total 6H), 1.30, 1.33, 1.36 and 1.39 (4 s, total6H), 0.95-1.06 (m, 6H).

Step 4 Synthesis of2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#112)

According to general procedure A, from #111 (210 mg, 0.230 mmol) indichloromethane (5 mL, 0.05 M) and diethylamine (5 mL) was synthesizedthe crude desired material, which was purified by silica gelchromatography (Gradient: 0% to 10% methanol in dichloromethane) to givea mixture of an oil and solid. Diethyl ether and heptane were added andthe mixture was concentrated in vacuo, producing #112 (81 mg, 51%) as awhite solid. LC-MS: m/z 690.4 [M+H⁺], retention time=1.10 minutes; HPLC(Protocol A): m/z 690.5 [M+H⁺], 712.4 [M+Na⁺], retention time=7.229minutes (purity >90%); ¹H NMR (400 MHz, DMSO-d₆), presumed to be amixture of rotamers, characteristic signals: δ [7.62 (br d, J=8 Hz),7.88 (br d, J=8 Hz), 8.07 (br d, J=9 Hz) and 8.11 (br d, J=9 Hz), total2H], 7.15-7.34 (m, 5H), [5.34 (d, J=4 Hz) and 5.41 (d, J=5 Hz), total1H], 3.18, 3.21, 3.23 and 3.25 (4 s, total 6H), 2.93 and 3.08 (2 br s,total 3H), 1.15, 1.18, 1.21 and 1.25 (4 s, total 6H).

Preparation ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (115)

Step 1 Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate(#113).

To a stirring mixture of dimer acid#5 (12.1 g, 23.0 mM) and #67 (11.5 g,23.0 mM) in 75 mL of dichloromethane under nitrogen, HATU (10.8 g, 27.6mM) was added followed by Hunig's base (12.1 mL, 69.0 mM). The reactionwas allowed to stir at room temperature for 15 hours. Reaction wasconcentrated to a smaller volume, taken up with ethyl acetate and washedwith 1 N HCl two times. The organic layer was then washed with brine,dried over sodium sulfate, filtered, and concentrated in vacuo. Residuewas then purified by silica gel chromatography (Gradient: 0% to 70%acetone in heptanes), producing #113 (12.3 g, 62%) as a white solid.LC-MS (Protocol Q): m/z 855.3 [M+H⁺], 877.2 [M+Na⁺], retention time=2.32minutes; HPLC (Protocol R): /z 855.5 [M+H⁺], retention time=9.596minutes (purity >97%).

Step 2 Synthesis of methylN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalaninate(#114)

According to general procedure A, from #113 (12 g, 14 mmol, 1 eq.),dichloromethane (60 mL, 0.24 M) and diethylamine (40 mL, 390 mM) wassynthesized #114 (5.9 g, 67%) white/slight yellow solid afterpurification by silica gel chromatography (Gradient: 0% to 25% methanolin dichloromethane). LC-MS (Protocal Q): m/z 633.0 [M+H⁺], retentiontime=1.19 minutes. HPLC (Protocol A): /z 633.5 [M+H⁺], retentiontime=7.142 minutes (purity >98%).

Step 3 Synthesis ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide-trifluoroaceticacid salt (#115)

To a stirring mixture ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanine (167 mg, 0.493mM), #114 (260 mg, 0.411 mM), and HATU (188 mg, 0.493 mM) in 10 mL ofdichloromethane, Hunig's base (0.14 mL, 0.82 mM) was added. The reactionwas allowed to stir at room temperature for 1 hour and 20 minutes.Reaction was reduced down. THF (9 mL) was added to crude material and tothis stirring mixture lithium hydroxide (49.2 mg, 2.06 mM) dissolved in3 mL of water was added. The reaction was allowed to stir at roomtemperature for 4 hours. Reaction was concentrated down followed bypurification by medium pressure reverse phase C18 chromatography(Gradient: 5% to 45% water in acetonitrile with 0.02% TFA in each phase)#115 (218 mg, 64%) white solid. LC-MS (Protocol Q): m/z 718.7 [M+H⁺],740.6 [M+Na⁺], retention time=1.21 minutes. HPLC (Protocol A at 45° C.):m/z 718.4 [M+H⁺], retention time=6.903 minutes. ¹H NMR (400 MHz,DMSO-d₆), δ 8.81-8.95 (m), 8.44-8.50 (m), 8.42 (d), 8.15 (d), 7.14-7.28(m), 4.71-4.78 (m), 4.57-4.66 (m), 4.49-4.56 (m), 4.41-4.48 (m),3.94-4.05 (m), 3.72-3.79 (m), 3.39-3.60 (m), 2.95-3.33 (m), 2.78-2.89(m), 2.69 (s), 2.43-2.50 (m), 2.08-2.42 (m), 1.60-1.92 (m), 1.20-1.57(m), 0.84-1.11 (m), 0.74-0.83 (m).

Preparation of2-methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#117) and2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#118)

Step 1 Synthesis of1-(tert-butoxycarbonyl)-2-methyl-L-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#116)

To a stirring solution of #114 (1.02 g, 1.61 mmol, 1.0 eq.) and1-(tert-butoxycarbonyl)-2-methyl-L-proline (443 mg, 1.93 mmol, 1.2 eq.)in 12 mL of dichloromethane, HATU (735 mg, 1.93 mmol, 1.2 eq.) was addedfollowed by Hunig's base (1.12 mL, 6.45 mmol, 4.0 eq.). The reaction wasallowed to stir at room temperature for 2 hours. The reaction wasreduced down, diluted with ethyl acetate before being washed with 0.5 NHCl and brine. Organics where then dried over sodium sulfate, reduced toa smaller volume, and then reduced down on silica. Silica chromatographywas then performed (Gradient: 0%-45% acetone in heptanes) producing #116(1.02 g, 74%) as a white solid. LC-MS (Protocol Q): m/z 844.3

[M+H⁺], 867.2 [M+Na⁺], retention time=2.15 minutes.

Step 2A Synthesis of2-methyl-L-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#117)

To a stirring solution of #116 (450 mg, 0.533 mmol, 1.0 eq.) in 7 mL ofdichloromethane at 0° C., TFA (3 mL, 40 mmol, 70 eq.) was added. Thereaction was allowed to stir at 0° C. for 5 minutes and then allowed towarm to room temperature while stirring for 20 minutes. Reaction wasreduced down, diluted with dichloromethane and a small amount ofmethanol before being reduced down onto silica. Silica chromatographywas then performed (Gradient: 0%-20% methanol in ethyl acetate)producing #117 (396 mg, 89%) as a white solid. LC-MS (Protocol Q): m/z744.5 [M+H⁺], 767.2 [M+Na⁺], retention time=1.40 minutes; HPLC (ProtocolA at 45° C.): m/z 744.5 [M+H⁺], retention time=7.149 minutes(purity >91%). ¹H NMR (400 MHz, DMSO-d₆), δ 8.73-9.14 (m), 8.66 (br d),8.50 (d), 8.22 (d), 7.12-7.25 (m), 4.67-4.74 (m), 4.41-4.63 (m),3.93-4.00 (m), 3.73 (dd), 3.63 (d), 3.46-3.57 (m), 3.38-3.45 (m),3.26-3.23 (m), 3.22-3.25 (m), 3.06-3.22 (m), 2.99-3.05 (m), 2.93-2.97(m), 2.80-2.89 (m), 2.75-2.78 (m), 2.64-2.67 (m), 2.46-2.50 (m),2.27-2.43 (m), 2.00-2.26 (m), 1.85-1.99 (m), 1.70-1.83 (m), 1.52-1.69(m), 1.33-1.51 (m), 1.18-1.31 (m), 0.98-1.07 (m), 0.93-0.97 (m),0.82-0.92 (m), 0.71-0.78 (m).

Step 2B Synthesis of2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#118)

To a stirring solution of #116 (435 mg, 0.515 mmol), in 4 mL of THFunder nitrogen, LiOH (24.7 mg, 1.03 mmol, 2.0 eq.) dissolved in 2 mL ofwater was added. The reaction was allowed to stir at room temperatureuntil LC-MS indicated saponification of methyl ester. Reaction wasconcentrated in vacuo and then placed underneath vacuum. Reaction wasdiluted with dichloromethane and placed underneath nitrogen. To thisstirring mixture TFA (3 mL, 40.5 mmol, 80 eq.) was added. Reaction wasallowed to stir at room temperature for 30 minutes. Reaction was thenreduced down. Residue was purified by medium pressure reverse phase C18chromatography (Gradient: 5% to 60% acetonitrile in water with 0.02% TFAin each phase) #118 (396 mg, 89%) as a white solid. LC-MS (Protocol Q):m/z 730.2 [M+H⁺], retention time=1.18 minutes; HPLC (Protocol A at 45°C.): m/z 730.5 [M+H⁺], retention time=7.088 minutes (purity >98%). ¹HNMR (400 MHz, DMSO-d₆), δ 9.04-9.13 (m), 8.75-8.87 (m), 8.70 (d), 8.38(d), 8.11 (d), 7.10-7.24 (m), 4.66-4.74 (m), 4.48-4.64 (m), 4.37-4.47(m), 3.91-3.99 (m), 3.77 (m), 3.47-3.56 (m), 3.33-3.47 (m), 3.08-3.30(m), 2.93-3.07 (m), 2.75-2.86 (m), 2.63-2.69 (m), 2.45-2.50 (m),2.28-2.44 (m), 2.03-2.27 (m), 1.88-2.02 (m), 1.68-1.86 (m), 1.55-1.67(m), 1.30-1.47 (m), 1.17-1.29 (m), 0.98-1.05 (m), 0.93-0.97 (m),0.83-0.92 (m), 0.71-0.79 (m).

Preparation of2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#123)

Step 1 Synthesis of(2R,3R)-3-{(2S)-1-[(9H-fluoren-9-ylmethoxy)carbonyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoicacid (#119)

To a stirring solution of #11 (2.4 g, 8.4 mmol, 1.0 eq.) in 10 mL ofdioxane under nitrogen, 4M HCl in dioxane (20 mL, 80 mM, 10 eq.) wasadded. The reaction was allowed to stir at room temperature for 3 hoursbefore being concentrated in vacuo and placed underneath high vacuum.Crude material was then dissolved with 30 mL of 10% Na₂CO₃. Thissolution was then added to a stirring solution of1-{[(9H-fluoren-9-ylmethoxy)carbonyl]oxy}pyrrolidine-2,5-dione (2.96 g,8.77 mmol, 1.05 eq.) in 30 mL of DME. Reaction was allowed to stir atroom temperature until TLC (20% methanol/40% ethyl acetate/40% heptanes)indicated the consumption of Boc de-protected starting material. Thereaction was concentrated in vacuo to a smaller volume, washed twicewith ether, acidified to pH 2 using concentrated HCl and then extractedthree times with a solution of 90% dichloromethane 10% methanol. Theorganics where washed with saturated sodium bicarbonate and brine beforebeing dried over sodium sulfate, filtered, and concentrated in vacuo toa brown solid #119 (3.4 g, quant.). LC-MS (Protocol Q): m/z 410.0[M+H⁺], retention time=1.81 minutes.

Step 2 Synthesis tert-butylN-[(2R,3R)-3-{(2S)-1[(9H-fluoren-9-ylmethoxy)carbonyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L-phenylalaninate(#120)

To a stirring solution of tert-butyl L-phenylalaninate, hydrochloridesalt (1.67 g, 6.5 mmol, 1.0 eq.) and #119 (5.9 g, 6.5 mmol, 1.0 eq.) in50 mL of dichloromethane and 5 mL of DMF, HATU (2.9 g, 7.9 mmol, 1.2eq.) was added followed by Hunig's base (5.6 mL, 32 mmol, 5.0 eq.). Thereaction was allowed to stir at room temperature for 45 minutes.Reaction was reduced down, diluted with ethyl acetate, washed with 0.5 NHCl and brine before being concentrated down onto silica. Silicachromatography was then performed (Gradient: 0%-25% acetone in heptane)producing #120 (3.14 g, 79%) as a white yellow solid. LC-MS (ProtocolQ): m/z 613.1 [M+H⁺] retention time=2.37 minutes.

Step 3 Synthesis of tert-butylN-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenylalaninate(#121)

To a stirring solution of #120 (2.87 g, 4.68 mmol, 1.00 eq.) in 20 mL ofdichloromethane, diethylamine (10 mL, 95 mM, 20.5 eq.) was added. Thereaction was allowed to stir at room temperature for 2 hours. Another(10 mL, 95 mmol, 20.5 eq.) of diethylamine was added and the reactionwas allowed to stir at room temperature for 3 more hours. Reaction wasconcentrated in vacuo and placed underneath high vacuum producing #121(1.8 g, quant.) yellow white oil solid mix. LC-MS (Protocol Q): m/z391.1 [M+H⁺] retention time=1.05 minutes.

Step 4 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#122)

To a stirring solution of #121 (0.55 g, 1.0 mmol, 1.0 eq.) in 10 mL ofdichloromethane and 1 mL of DMF, #32 (0.62 g, 1.0 mmol, 1.0 eq.) wasadded followed by HATU (0.42 g, 1.1 mmol, 1.1 eq.) and Hunig's base(0.72 mL, 4.1 mmol, 4.0 eq.). The reaction was allowed to stir at roomtemperature for approximately 21 hours. Reaction was reduced down,diluted with ethyl acetate, and then washed with 0.5 N HCl and brine.Organic layer was dried over sodium sulfate, filtered, and concentratedto a smaller volume before being concentrated down onto silica. Silicachromatography was then performed (Gradient: 0%-40% acetone in heptane)producing #122 (0.62 g, 62%) as a white solid. LC-MS (Protocol Q): m/z982.3 [M+H⁺] retention time=2.44 minutes.

Step 5 Synthesis2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#123)

To a stirring mixture of #122 (600 mg, 0.611 mmol, 1.00 eq.) in 15 mL ofdichloromethane, diethylamine (5 mL, 50 mmol, 80 eq.) was added. Thereaction was allowed to stir at room temperature for 3 hours. Reactionwas concentrated in vacuo and mixture was purified by SilicaChromatography (Gradient: 0%-40% methanol in dichloromethane) producing#123 (0.46 g, 99%) as a solid. LC-MS (Protocol Q1): m/z 760.3 [M+H⁺]retention time=0.83 minutes. ¹H NMR (400 MHz, CD₃OD), δ 7.14-7.30 (m),4.70-4.78 (m), 4.56-4.64 (m), 4.05-4.19 (m), 3.87 (dd), 3.79-3.84 (m),3.72-3.77 (m), 3.62-3.70 (m), 3.46-3.56 (m), 3.37-3.45 (m), 3.33-3.36(m), 3.16-3.24 (m), 3.09-3.11 (m), 2.98-3.05 (m), 2.95 (d), 2.91 (d),2.87 (d), 2.83 (d),2.73-2.79 (m), 2.40-2.51 (m), 2.29-2.39 (m),2.16-2.28 (m), 2.04-2.15 (m), 2.01 (s), 1.73-1.96 (m), 1.50-1.68 (m),1.47-1.49 (m), 1.46 (s), 1.43 (s), 1.38 (s), 1.35 (d), 1.23-1.32 (m),1.17-1.22 (m), 1.15 (d), 1.04-1.11 (m), 0.94-1.03 (m), 0.82-0.91 (m).

Preparation of methylN-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-(3-amino-2,2-dimethylpropanoyl)-L-valyl](methyl)amino}-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoylpL-phenylalaninate(#126)

Step 1 Synthesis of3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-2,2-dimethylpropanoic acid(#124)

A solution of 3-amino-2,2-dimethylpropanoic acid hydrochloride (1.0 g,6.5 mmol, 1.0 eq.) in 10 mL of 10% Na₂CO₃ was added to a solution of1-{[(9H-fluoren-9-ylmethoxy)carbonyl]oxy}pyrrolidine-2,5-dione (2.3 g,6.5 mmol, 1.0 eq.) in 10 mL of DME. The reaction was allowed to stir atroom temperature overnight. Reaction was concentrated to a smallervolume and then washed two times with ether. The aqueous layer wasacidified to pH<2 with concentrated HCl and then extracted three timeswith a 10% methanol 90% dichloromethane solution. The organics wherecombined before being washed with 1M HCl and brine. The organic layerwas dried over sodium sulfate and concentrated in vacuo producing #124(2.2 g, 98%) as a white solid. LC-MS (Protocol Q1): m/z 362.0 [M+Na⁺]retention time=0.89 minutes.

Step 2 Synthesis of methylN-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-2,2-dimethylpropanoyl)-L-valyl](methyl)amino}-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L-phenylalaninate(#125)

To a stirring solution of #114 (200 mg, 0.316 mmol, 1.00 eq.) in 2 mL ofdichloromethane, #124 (107 mg, 0.316 mmol, 1.00 eq.) was added followedby Hunig's base (0.167 mL, 0.948 mmol, 3.00 eq.) and HATU (149 mg, 0.379mmol, 1.20 eq.). The reaction was allowed to stir at room temperaturefor ˜12 hours. The reaction was concentrated to a smaller volume, takenup in 10 mL of ethyl acetate, and washed two times with 5 mL of 1M HCl,and once with 5 mL of brine. The organic layer was dried over sodiumsulfate and decanted. Organics where concentrated in vacuo and the crudematerial was taken up in dichloromethane. The precipitate was filtered.The organic layer was concentrated in vacuo and the residue was purifiedby silica chromatography (Gradient: 0%-50% acetone in heptane) producing#125 (235 mg, 78%) as a white solid. LC-MS (Protocol Q): m/z 954.2[M+H⁺] retention time=2.28 minutes.

Step 3 Synthesis of methylN-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-(3-amino-2,2-dimethylpropanoyl)-L-valyl](methyl)amino}-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L-phenylalaninate(#126)

To a stirring solution of #125 (235 mg, 0.246 mmol, 1.00 eq.) in 2 mL ofTHF, (1 mL, 10 mM, 40.6 eq.) of diethylamine was added. The reaction wasallowed to stir at room temperature for 3 hours. Reaction wasconcentrated in vacuo and the residue was purified by silicachromatography (Gradient: 0%-30% methanol in ethyl acetate) producing#126 (101 mg, 56%) as a white solid. LC-MS (Protocol Q): m/z 732.2[M+H⁺] retention time=1.32 minutes. ¹H NMR (400 MHz, DMSO-d₆), δ 8.51(dd), 8.28 (d), 7.15-7.29 (m), 5.77 (s), 4.55-4.77 (m), 4.44-4.54 (m),3.94-4.10 (m), 3.73-3.79 (m), 3.66 (d), 3.49-3.60 (m), 3.40-3.48 (m),3.10-3.36 (m), 3.00-3.09 (m), 2.83-2.98 (m), 2.57-2.77 (m), 2.19-2.46(m), 1.87-2.14 (m), 1.61-1.86 (m), 1.36-1.55 (m), 1.23-1.36 (m),1.12-1.22 (m), 0.97-1.11 (m), 0.82-0.96 (m), 0.73-0.81 (m).

Preparation ofN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#130)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#127)

To a round bottom flask containing #6 (4.7 g, 7.9 mmol, 1.0 eq.) andN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanine (3.2 g, 9.4mmol, 1.2 eq.) and a stir bar under nitrogen, 50 mL of dichloromethanewas added followed by HATU (3.6 g, 9.4 mmol, 1.2 eq.) and Hunig's base(5.5 mL, 32 mmol, 4.0 eq.). The reaction was allowed to stir at roomtemperature for ˜12 hours. Reaction was reduced to a smaller volume,taken up in ethyl acetate, before being washed with 1 N HCl, and brine.Organics where then dried over sodium sulfate, filtered and then reduceddown onto silica. Residue was purified by Silica Chromatography(Gradient: 0%-30% acetone in heptane) producing #127 (4.2 g, 78%) as awhite solid. LC-MS (Protocol Q): m/z 680.2 [M+H⁺] retention time=2.52minutes.

Step 2 SynthesisN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide,(#128)

To a stirring solution of #127 (4.2 g, 6.1 mmol, 1.0 eq.) in 21 mL ofdichloromethane under nitrogen, (7 mL, 90 mmol, 10 eq.) of TFA wasadded. The reaction was allowed to stir at room temperature for −4hours. Reaction was concentrated in vacuo, azeotroped once with heptane,and then placed underneath high vacuum yielding #128 as a white slightyellow solid (3.8 g, quant.). LC-MS (Protocol Q): m/z 624.2 [M+H⁺]retention time=2.01 minutes.

Step 3 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#129)

To a stirring solution of #128 (1.67 g, 3.1 mmol, 1.0 eq.) in 20 ofdichloromethane and 2 mL of DMF, #121 (2.4 g, 3.1 mmol, 1.0 eq.) wasadded followed by HATU (1.29 g, 3.39 mmol, 1.1 eq.) and then Hunig'sbase (2.2 mL, 12.3 mmol, 4.0 eq.). The reaction was allowed to stir atroom temperature for -2 hours. Reaction was reduced down, diluted withethyl acetate before being washed with 0.5 N HCl and brine. Organicswhere dried over sodium sulfate and then reduced down onto silica.Residue was purified by Silica Chromatography (Gradient: 0%-50% acetonein heptanes) producing #129 (1.9 g, 62%) as a white solid. LC-MS(Protocol Q): m/z 996.3 [M+H⁺] retention time=2.53 minutes.

Step 4 Synthesis ofN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#130)

To a stirring solution of #129 (823 mg, 0.826 mmol, 1.00 eq.) in 15 mLof dichloromethane, diethylamine (4 mL, 40 mmol, 50 eq.) was added. Thereaction was allowed to stir at room temperature for ˜14½ hours. Thereaction was concentrated in vacuo and azeotroped once with heptanes.Residue with diluted with dichloromethane and a small amount of methanolbefore being reduced down onto silica. Residue was purified by SilicaChromatography (Gradient: 0%-20% methanol in ethyl acetate) producing#130 (518 mg, 81%) as a white solid. LC-MS (Protocol Q): m/z 774.3[M+H⁺] retention time=1.48 minutes. HPLC (Protocol A at 25° C.): m/z774.5 [M+H⁺], retention time=7.733 minutes (purity >98%). ¹H NMR (400MHz, DMSO-d₆), δ 8.36 (d). 8.14 (d), 7.81 (t), 7.14-7.25 (m), 7.01-7.07(m), 4.87-4.94 (m), 4.78-4.85 (m), 4.67-4.76 (m), 4.46-4.65 (m),4.29-4.40 (m), 3.93-4.03 (m), 3.70-3.81 (m), 3.49-3.60 (m), 3.38-3.47(m), 3.29-3.36 (m), 3.15-3.28 (m), 2.98-3.13 (m), 2.94 (br s), 2.74-2.89(m), 2.64-2.69 (m), 2.18-2.45 (m), 2.02-2.14 (m), 1.90-2.01 (m),1.62-1.87 (m), 1.40-1.55 (m), 1.37 (d), 1.20-1.33 (m), 1.16 (d),1.01-1.10 (m), 0.90-0.98 (m), 0.82-0.89 (m), 0.69-0.79 (m).

Preparation of2-methyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#131)

Step 1 Synthesis of2-methyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamidetrifluoroacetic acid salt (#131)

To a stirring solution of #114 (164 mg, 0.259 mmol, 1.0 eq.) and1-(tert-butoxycarbonyl)-2-methyl-D-proline (71.3 mg, 0.311 mmol, 1.2eq.) in 4 mL of dichloromethane, HATU (118 mg, 0.311 mmol, 1.2 eq.) wasadded followed by Hunig's base (0.180 mL, 1.04 mmol, 4 eq.). Thereaction was allowed to stir at room temperature for -30 minutes.Reaction was reduced down. Reaction was taken up in 3.5 mL ofdichloromethane and placed under nitrogen. To this stirring solution,TFA (1.5 mL, 20 mmol, 76 eq.) was added. The reaction was allowed tostir at room temperature for -1 hour. Reaction was reduced down andplaced underneath high vacuum. Purification by (Method J*) affords #131(119 mg, 54%) as a white solid. HPLC (Protocol A at 45° C.): m/z 744.5[M+H⁺], retention time=7.342 minutes (purity >98%). ¹H NMR (400 MHz,DMSO-d₆), δ 9.08-9.18 (m), 8.79-8.89 (m), 8.76 (t), 8.54 (d), 8.29 (d),7.14-7.31 (m), 4.70-4.79 (m), 4.57-4.66 (m), 4.45-4.55 (m), 3.96-4.04(m), 3.74-3.80, 3.66 (d), 3.48-3.61 (m), 3.40-3.48 (m), 3.09-3.34 (m),3.00-3.09 (m), 2.95-3.00 (m), 2.83-2.93 (m), 2.36-2.53 (m), 2.21-2.35(m), 2.10-2.19 (m), 1.99-2.10 (m), 1.61-1.09 (m), 1.36-1.53 (m),1.21-1.35 (m), 1.02-1.10 (m), 0.94-1.0 (m), 0.86-0.93 (m), 0.73-0.82(m).

Preparation of2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#134)

Step 1 Synthesis ofN˜2˜-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#132)

To a flask containing #@5 (1.14 g, 2.17 mmol, 1.0 eq.), 10 mL ofdichloromethane was added followed by Hunig's base (1.15 mL, 6.52 mmol,3.0 eq.), HATU (1.02 g, 2.61 mmol, 1.2 eq.), and #110 (0.776 g, 2.17mmol, 1.0 eq.). Reaction was allowed to stir at room temperature for 30minutes and then concentrated in vacuo. Crude material was taken up in50 mL of ethyl acetate, washed two times with 25 mL of 1 M HCl, and oncewith 25 mL of brine. Organics where dried over sodium sulfate anddecanted. Organics where concentrated in vacuo, taken up in 30 mL ofdichloromethane, and the resulting precipitate was filtered off Organicswhere concentrated in vacuo and the residue was purified by silicachromatography (Gradient: 0%-50% acetone in heptanes) producing #132(1.33 g, 81%) as a solid. LC-MS (Protocol Q): m/z 849.2 [M+Na⁺]retention time=2.19 minutes.

Step 2 Synthesis ofN-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#133)

To a stirring solution of #132 (1.33 g, 1.60 mmol, 1.0 eq) in 10 mL ofTHF diethylamine (5 mL, 50 mM, 31.3 eq) was added. The reaction wasallowed to stir at room temperature for 4 hours. Reaction wasconcentrated in vacuo and the residue was purified by silicachromatography (Gradient: 0%-30% methanol in ethyl acetate) producing#133 (418 mg, 43%) as a white solid. LC-MS (Protocol Q1): m/z 605.2[M+H⁺] retention time=1.48 minutes.

Step 3 Synthesis of2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#134)

HATU (151 mg, 0.398 mmol, 1.2 eq), #133 (201 mg, 0.332 mmol, 1.0 eq.)and 1-(tert-butoxycarbonyl)-2-methyl-L-proline (91.3 mg, 0.398 mM, 1.2eq.) where combined in a round bottom flask containing a stir bar undernitrogen. 5 mL of dichloromethane was added followed by Hunig's base(0.231 mL, 1.33 mmol, 4.0 eq.). The reaction was allowed to stir at roomtemperature for -15 hours. Reaction was then concentrated in vacuo andplaced underneath high vacuum. 4 mL of dioxane was then added to theresidue followed by 4M HCl in dioxane (4 mL, 20 mmol, 50 eq.). Reactionwas then allowed to stir at room temperature for 1 hour. Reaction wasthen concentrated in vacuo and the residue was purified by mediumpressure reverse phase C18 chromatography (Gradient: 5% to 90%acetonitrile in water with 0.02% TFA in each phase) #134 (237 mg, 86%)as a white solid. LC-MS (Protocol Q): m/z 716.3 [M+H⁺], retentiontime=1.16 minutes; HPLC (Protocol A at 45° C.): /z 716.5 [M+H⁺],retention time=6.930 minutes (purity >98%). ¹H NMR (400 MHz, DMSO-d₆),9.12-9.21 (m), 8.79-8.90 (m), 8.70-8.78 (m), 7.95 (d), 7.64 (d),7.25-7.36 (m), 7.16-7.23 (m), 4.74-4.80 (m), 4.61-4.69 (m), 4.41-4.59(m), 3.91-4.06 (m), 3.78 (dd), 3.54-3.64 (m), 3.45-3.51 (m), 3.17-3.36(m), 3.02-3.15 (m), 3.00 (br s), 2.40-2.48 (m), 2.24-2.35 (m), 1.91-2.21(m), 1.68-1.90 (m), 1.61-1.68 (m), 1.48-1.59 (m), 1.22-1.35 (m),0.97-1.09 (m), 0.84-0.97 (m), 0.74-0.83 (m).

Preparation ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(methylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#140),N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-2-amino-1-benzyl-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#141),N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-oxo-2-(propylamino)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#142),N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(diethylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#143), andN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(tert-butylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#144)

Step 1 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-5-methyl-1-oxo-1-(pentafluorophenoxy)heptan-4-yl]-N-methyl-L-valinamide(#135)

Pentafluorophenyl 3,3,3-trifluoropropanoate (2.44 mL, 13.4 mmol, 2.0eq.) was added to a solution of #128 (4.18 g, 6.70 mmol, 1.0 eq.) in 50mL of dichloromethane followed by pyridine (1.61 mL, 20.1 mmol, 3.0eq.). Reaction was allowed to stir at room temperature for -12 hours.Reaction was concentrated in vacuo and the residue was purified bysilica chromatography (Gradient: 0%-70% acetone in heptanes) producing#135 (5.2 g, 98%) as a white foam. LC-MS (Protocol Q1): m/z 812.1[M+Na⁺] retention time=1.24 minutes.

Step 2 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#136)

To a stirring solution of 4M HCl in dioxane (10 mL, 25 mmol, 3.7 eq.) in10 mL of dioxane #11 (2.31 g, 8.05 mmol, 1.2 eq.) was added. Thereaction was allowed to stir at room temperature for 6 hours. Thereaction was concentrated in vacuo producing a yellow gum. A solution of#135 (5.3 g, 6.7 mmol, 1.0 eq.) in 30 mL of dichloromethane was added tothe previous residue followed by Hunig's base (3.5 mL, 20 mmol, 3 eq.).The reaction was allowed to stir at room temperature for 4 hours. Thereaction was diluted with dichloromethane before being washed with a 1%HCl aqueous solution and then brine. The organics layer was dried oversodium sulfate, concentrated in vacuo, and the residue was purified bysilica chromatography (Gradient: 20%-50% ethyl acetate in heptanesfollowed by 93% ethyl acetate 6.6% methanol and 0.4% acetic acid)producing #136 (4.87 g, 92%) as a off white solid. LC-MS (Protocol Q1):m/z 793.3 [M+H⁺] retention time=1.07 minutes.

Step 3 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-(pentafluorophenoxy)propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#137)

Pentafluorophenyl 3,3,3-trifluoropropanoate (1.3 mL, 7.1 mmol, 2.0 eq.)was added to a solution of #136 (2.8 g, 3.5 mmol, 1.0 eq.) in 30 mL ofdichloromethane followed by the addition of pyridine (0.85 mL, 10.6 mM).The reaction was allowed to stir at room temperature for 2 hours. Thereaction was concentrated in vacuo, and the residue was purified bysilica chromatography (Gradient: 0%-70% acetone in heptane) producing#137 (3.1 g, 92%) as a white powder. LC-MS (Protocol Q1): m/z 959.2[M+H⁺] retention time=1.28 minutes.

Step 4 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#138)

To a stirring solution of #137 (493 mg, 0.514 mmol, 1.0 eq.) in 4 mL ofDMF, L-phenylalanine (84.9 mg, 0.514 mmol, 1.0 eq) was added followed byHunig's base (0.27 mL, 1.54 mmol, 3.0 eq.). The reaction was allowed tostir at room temperature for ˜12 hours. Reaction was concentrated invacuo and residue was purified by silica chromatography (Gradient:0%-100% ethyl acetate in heptane) producing #138 (200 mg, 41%) as awhite foam. LC-MS (Protocol Q1): m/z 940.3 [M+H⁺] retention time=1.08minutes.

Step 5 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S)-1-oxo-1-(pentafluorophenoxy)-3-phenylpropan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#139)

To a stirring solution of #138 (200 mg, 0.213 mmol, 1.0 eq.) in 5 mL ofdichloromethane, Pentafluorophenyl 3,3,3-trifluoropropanoate (126 mg,0.426 mM, 2.0 eq.) was added followed by pyridine (0.051 mL, 0.64 mmol,3.0 eq.). The reaction was allowed to stir at room temperature for ˜12hours. Reaction was concentrated in vacuo and the residue was purifiedby silica chromatography (Gradient: 0%-100% ethyl acetate in heptanes)producing #139 (174 mg, 74%) as a yellow oil. LC-MS (Protocol Q1): m/z1128 [M+Na⁺] retention time=1.23 minutes.

Step 6A Synthesis ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(methylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#140)

To a stirred solution of #139 (20 mg, 0.018 mmol, 1.0 eq.) in 1 mL ofTHF methylamine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) was added, andthe mixture was stirred at room temperature for 3 hours. The reactionwas reduced down, and diluted with dmso, and subjected to purification(Method J*). The fractions were collected and concentrated in vacuo togive #140 (4.0 mg, 30%) as a white solid. LC-MS (Protocol Q1): m/z 731.2[M+H⁺], retention time=0.70 minutes. ¹H NMR (400 MHz, methanol-d₄),7.30-7.41 (m), 4.71-4.78 (m), 4.58-4.69 (m), 4.04-4.15 (m), 3.86-3.98(m), 3.73-3.78 (m), 3.61-3.70 (m), 3.50-3.58 (m), 3.32-3.47 (m),3.23-3.26 (m), 3.17-3.22 (m), 3.07-3.15 (m), 2.95-2.98 (m), 2.76-2.91(m), 2.68-2.75 (m), 2.63-2.66 (m), 2.43-2.51 (m), 2.22-2.28 (m),1.99-2.11 (m), 1.74-1.96 (m), 1.21-1.31 (m), 1.17-1.20 (m), 0.92-1.10(m), 0.79-0.89 (m).

Step 6B Synthesis ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-2-amino-1-benzyl-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#141)

Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0eq.), ammonia solution (7M in methanol, 0.026 mL, 0.18 mmol, 10 eq.) andpurification (Method J*), #141 (3.0 mg, 20%) was obtained as a whitesolid. LC-MS (Protocol Q): m/z 717.2 [M+H⁺], retention time=0.79minutes. ¹H NMR (400 MHz, methanol-d₄), 7.22-7.30 (m), 7.14-7.21 (m),4.57-4.4.80 (m), 4.02-4.17 (m), 3.92-3.98 (m), 3.84-3.91 (m), 3.32-3.74(m), 3.17-3.27 (m), 3.06-3.14 (m), 2.77-3.05 (m), 2.65 (s), 2.43-2.51(m), 2.21-2.26 (m), 1.98-2.13 (m), 1.70-1.94 (m), 1.32-1.69 (m),1.16-1.31 (m), 0.89-1.13 (m), 0.80-0.88 (m).

Step 6C Synthesis ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-oxo-2-(propylamino)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#142)

Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0eq.) n-propylamine (1M in THF, 0.18 mL, 0.18 mmol, 10. eq) andpurification (Method J*), #142 (3.0 mg, 20%) was obtained as a whitesolid. LC-MS (Protocol Q): m/z 759.2 [M+H⁺], retention time=0.74minutes. ¹H NMR (400 MHz, methanol-d₄), 7.15-7.29 (m), 4.71-4.79 (m),4.52-4.68 (m), 4.04-4.17 (m), 3.87-3.99 (m), 3.73-3.99 (m), 3.73-3.79(m), 3.50-3.70 (m), 3.34-3.49 (m), 3.06-3.23 (m), 2.79-2.99 (m),2.44-2.50 (m), 2.28-2.43 (m), 2.22-2.27 (m), 1.75-2.10 (m), 1.34-1.61(m), 1.16-1.29 (m), 0.92-1.10 (m), 0.77-0.89 (m).

Step 6D Synthesis ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(diethylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#143)

Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0eq.) diethylamine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) andpurification (Method J*), #143 (4.0 mg, 30%) was obtained as a whitesolid. LC-MS (Protocol Q): m/z 773.3 [M+H⁺], retention time=0.77minutes. ¹H NMR (400 MHz, methanol-d₄), 7.16-7.33 (m), 5.10-5.17 (m),4.96-5.07 (m), 4.68-4.75 (m), 4.60-4.65 (m), 3.61-4.23 (m), 3.35-3.67(m), 3.16-3.26 (m), 2.99-3.15 (m), 2.78-2.94 (m), 2.30-2.52 (m),2.19-2.28 (m), 1.73-2.13 (m), 1.83-1.45 (m), 1.19-1.31 (m), 0.92-1.18(m), 0.80-0.89 (m).

Step 6E Synthesis ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-(tert-butylamino)-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt #144

Following the same procedure as #140 using #139 (20 mg, 0.018 mmol, 1.0eq.) tert-butylamine (1M in THF, 0.18 mL, 0.18 mmol, 10 eq.) andpurification (Method J*), #144 (3.4 mg, 24%) was obtained as a whitesolid. LC-MS (Protocol Q1): m/z 773.3 [M+H⁺], retention time=0.74minutes. ¹H NMR (400 MHz, DMSO-d₆), δ 8.21 (d), 8.03-7.98 (m), 7.92 (d),7.81-7.62 (m), 7.46-7.16 (m), 4.83-4.69 (m), 4.68-4.56 (m), 4.21-4.07(m), 3.92-3.86 (m), 3.83-3.80 (m), 3.74-3.65 (m), 3.60-3.48 (m),3.47-3.36 (m), 3.28-3.13 (m), 3.11-3.01 (m), 2.96-2.82 (m), 2.69-2.62(m), 2.54-2.43 (m), 2.38-2.12 (m), 2.00-1.76 (m), 1.69-1.161 (m),1.60-1.53 (m), 1.52-0.98 (m), 0.94-0.86 (m).

Preparation ofN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#145)

Step 1 Synthesis ofN,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#145)

To a stirring solution of #137 (300 mg, 0.313 mmol, 1.0 eq.) in 3 mL ofDMF, (1S,2R)-2-amino-1-phenylpropan-1-ol (54.8 mg, 0.344 mmol, 1.1 eq.)was added followed by Hunig's base (0.164 mL, 0.939 mmol, 3.0 eq). Thereaction was allowed to stir at room temperature for ˜12 hours.Piperidine 20% solution in DMF (1 mL, 2.2 mmol, 7.0 eq.) was then addedand the reaction was allowed to stir at room temperature for 2 hours.Purification (Method J*) followed by concentration of appropriate testtubes produced #145 (190 mg, 74%) as a white powder. LC-MS (Protocol Q):m/z 704.3 [M+H⁺], retention time=0.67 minutes. ¹H NMR (400 MHz, CD₃OD),δ 7.97 (d), 7.73 (d), 7.37-7.41 (m), 7.27-7.36 (m), 7.19-7.25 (m),4.70-4.75 (m), 4.58-4.63 (m), 4.49-4.54 (m), 4.14-4.30 (m), 4.04-4.11(m), 3.87 (dd), 3.63-3.77 (m), 3.51-3.58 (m), 3.46-3.49 (m), 3.38-3.43(m), 3.25-3.37 (m), 3.15-3.23 (m), 3.11-3.14 (m), 3.01-3.02 (m),2.59-2.64 (m), 2.52-2.55 (m), 2.44-2.52 (m), 2.41-2.43 (m), 2.07-2.26(m), 1.73-2.0 (m), 1.65-1.73 (m), 1.59-1.65 (m), 1.51-1.59 (m),1.32-1.46 (m), 1.23-1.26 (m), 1.08-1.21 (m), 0.94-1.07 (m), 0.83-0.92(m).

Preparation of3-methyl-D-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#146),3-methyl-L-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#147),L-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#148), andD-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#149)

Step 1A Synthesis of3-methyl-D-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#146)

A solution of #114 (225 mg, 0.356 mmol, 1.0 eq.) in 2 mL ofdichloromethane was added to a solution ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-3-methyl-D-isovaline (126 mg, 0.356mmol, 1.0 eq.) in 4 mL of dichloromethane. Hunig's base (0.188 mL, 1.07mmol, 3.0 eq.) was added followed by HATU (167 mg, 0.427 mmol, 1.2 eq).The reaction was allowed to stir at room temperature for 12 hours. Thereaction was concentrated in vacuo and then taken up in ethyl acetatebefore being washed two times with 1M HCl and once with brine. Theorganic layer was dried over sodium sulfate and decanted. The organicsolvent was removed in a genevac. THF (4 mL) was added followeddiethylamine (2 mL, 19 mmol, 53.4 eq.). The reaction was allowed to stirfor ˜12 hours. Reaction was concentrated using a genevac followed bysilica chromatography (Gradient: 0%-30% methanol in ethyl acetate)producing #146 (183 mg, 69%) as a solid. LC-MS (Protocol Q): m/z 746.4[M+H⁺] retention time=1.37 minutes. ¹H NMR (400 MHz, DMSO-d₆), δ 8.55(d), 8.26-8.36 (m), 7.88-8.03 (m), 7.81 (d), 7.41-7.53 (m), 7.13-7.30(m), 7.01 (s), 4.71-4.79 (m), 4.44-4.70 (m), 3.96-4.04 (m), 3.70-3.80(m), 3.62-3.69 (m), 3.40-3.61 (m), 2.76-3.35 (m), 2.67-2.71 (m),2.56-2.58 (m), 2.06-2.46 (m), 1.61-1.90 (m), 1.14-1.54 (m), 0.72-1.12(m).

Step 1B Synthesis of3-methyl-L-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide(#147)

A solution of #114 (224 mg, 0.354 mmol, 1.0 eq.) in 2 mL ofdichloromethane was added to a solution ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-3-methyl-L-isovaline (125 mg, 0.354mmol, 1.0 eq.) in 4 mL of dichloromethane. Hunig's base (0.187 mL, 1.06mmol, 3.0 eq.) was added followed by HATU (167 mg, 0.425 mmol, 1.2 eq.).The reaction was allowed to stir at room temperature for 12 hours. Thereaction was concentrated in vacuo and then taken up in ethyl acetatebefore being washed two times with 1M HCl and once with brine. Theorganic layer was dried over sodium sulfate and decanted. The organicsolvent was removed in a genevac. THF (4 mL) was added followeddiethylamine (2 mL, 19 mmol, 53.7 eq.). The reaction was allowed to stirfor ˜12 hours. Reaction was concentrated using a genevac followed bysilica chromatography (Gradient: 0%-30% methanol in ethyl acetate)producing #147 (216 mg, 82%) as a solid. LC-MS (Protocol Q): m/z 746.6[M+H⁺] retention time=1.29 minutes. ¹H NMR (400 MHz, DMSO-d₆), δ 8.56(m), 8.31-8.39 (m), 8.50 (d), 8.30 (br d), 7.87-8.01 (m), 7.80 (d),7.40-7.53 (m), 7.14-7.30 (m), 4.45-4.78 (m), 3.94-4.04 (m), 3.70-3.79(m), 3.61-3.69 (m), 3.42-3.59 (m), 2.97-3.37 (m), 2.80-2.92 (m),2.32-2.49 (m), 2.05-2.30 (m), 1.61-1.89 (m), 1.37-1.56 (m), 1.14-1.135(m), 0.70-1.11 (m).

Step 1C Synthesis ofL-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#148)

A solution of #114 (447 mg, 0.707 mmol, 1.0 eq.) in 2 mL ofdichloromethane was added to a solution ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-isovaline (240 mg, 0.707 mmol,1.0 eq.) in 4 mL of dichloromethane. Hunig's base (0.373 mL, 2.12 mmol,3.0 eq.) was added followed by HATU (332 mg, 0.425 mmol, 1.2 eq.). Thereaction was allowed to stir at room temperature for 12 hours. Thereaction was concentrated in vacuo and then taken up in ethyl acetatebefore being washed two times with 1M HCl and once with brine. Theorganic layer was dried over sodium sulfate and decanted. The organicsolvent was removed in a genevac. THF (4 mL) was added followed bydiethylamine (2 mL, 19 mmol, 26.9 eq.). The reaction was allowed to stirfor ˜12 hours. Reaction was concentrated using a genevac followed bysilica chromatography (Gradient: 0%-30% methanol in ethyl acetate)producing #148 (182 mg, 35%) as a solid. LC-MS (Protocol Q1): m/z 732.3[M+H⁺] retention time=0.71 minutes. ¹H NMR (400 MHz, DMSO-d₆), δ 8.56(d), 8.46-8.52 (m), 8.30 (d), 8.02-8.15 (m), 7.98 (d), 7.80 (d),7.40-7.53 (m), 7.15-7.30 (m), 4.70-4.80 (m), 4.44-4.69 (m), 3.96-4.05(m), 3.70-3.79 (m), 3.62-3.69 (m), 3.41-3.59 (m), 2.99-3.35 (m),2.31-2.95 (m), 2.67-2.71 (m), 2.55-2.59 (m), 2.32-2.48 (m), 2.20-2.31(m), 1.97-2.19 (m), 1.61-1.88 (m), 1.37-1.56 (m), 1.20-1.34 (m),1.14-1.19 (m), 1.02-1.11 (m), 0.97-1.01 (m), 0.86-0.96 (m), 0.71-0.83(m).

Step 1D Synthesis ofD-isovalyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#149)

A solution of #114 (447 mg, 0.707 mmol, 1.0 eq.) in 2 mL ofdichloromethane was added to a solution ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-isovaline (240 mg, 0.707 mmol,1.0 eq.) in 4 mL of dichloromethane. Hunig's base (0.373 mL, 2.12 mmol,3 eq.) was added followed by HATU (332 mg, 0.425 mmol, 1.2 eq.). Thereaction was allowed to stir at room temperature for 12 hours. Thereaction was concentrated in vacuo and then taken up in ethyl acetatebefore being washed two times with 1M HCl and once with brine. Theorganic layer was dried over sodium sulfate and decanted. The organicsolvent was removed in a genevac. THF (4 mL) was added followed bydiethylamine (2 mL, 19 mmol, 26.9 eq.). The reaction was allowed to stirfor ˜12 hours. Reaction was concentrated using a genevac followed bysilica chromatography (Gradient: 0%-30% methanol in ethyl acetate)producing #149 (154 mg, 30%) as a solid. LC-MS (Protocol Q): m/z 732.0[M+H⁺] retention time=1.24 minutes. ¹H NMR (400 MHz, DMSO-d₆), δ 8.55(d), 8.38-8.46 (m), 8.29 (d), 8.03-8.14 (m), 7.97 (d), 7.81 (d),7.40-7.53 (m), 7.14-7.28 (m), 7.02 (s), 4.71-4.79 (m), 4.43-4.69 (m),3.96-4.05 (m), 3.71-3.80 (m), 3.62-3.70 (m), 3.49-3.60 (m), 3.40-3.48(m), 3.15-3.34 (m), 3.10-3.14 (m), 3.01-3.09 (m), 2.94-3.00 (m),2.83-2.93 (m), 2.65-2.71 (m), 2.55-2.59 (m), 2.32-2.48 (m), 2.04-2.31(m), 1.61-1.89 (m), 1.37-1.52 (m), 1.21-1.35 (m), 1.15-1.20 (m),1.02-1.10 (m), 0.75-1.01 (m).

Preparation of1,2-dimethyl-L-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#151)

Step 1 Synthesis of 1,2-dimethyl-L-proline (#150)

A parr flask containing 2-methyl-L-proline (1.0 g, 7.7 mmol, 1.0 eq.),40 mL of methanol, Formaldehyde 37 wt. % in water (2.1 mL, 77 mmol, 10eq.), and Palladium 10 wt. % on Carbon (313 mg, 2.94 mmol, 0.38 eq.) wasplaced on a parr shaker and allowed to shake under 40 psi of hydrogenfor -12 hours. Hydrogen was removed and the reaction was filteredthrough a pad of celite which was rinsed with a solution of 50% methanol50% dichloromethane. Residue was concentrated in vacuo yielding #150(1.1 g, 100%) as a white slight black colored solid. LC-MS (Protocol Q):m/z 144.0 [M+H⁺] retention time=0.17 minutes.

Step 2 Synthesis of1,2-dimethyl-L-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#151)

To a stirring mixture of #114 (125 mg, 0.198 mmol, 1.0 eq), #150 (37 mg,0.26 mmol, 1.3 eq.), and HATU (98 mg, 0.26 mmol, 1.3 eq.) in 5 mL ofdichloromethane, Hunig's base (0.14 mL, 0.80 mmol, 4.1 eq.) was added.The reaction was allowed to stir at room temperature for 1 hour.Reaction was concentrated in vacuo. THF (6 mL) was added to crudematerial. To this stirring mixture LiOH (14 mg, 0.59 mmol, 3.0 eq)dissolved in 2 mL of water was added. Reaction was allowed to stir atroom temperature for 90 minutes. Reaction was concentrated in vacuo andresidue was purified by medium pressure reverse phase C18 chromatography(Gradient: 5% to 40% acetonitrile in water with 0.02% TFA in each phase)#151 (147 mg, 69%) as a white solid. LC-MS (Protocol Q): m/z 744.3[M+H⁺], retention time=1.19 minutes; HPLC (Protocol A at 45° C.): m/z744.4 [M+H⁺], retention time=6.631 minutes (purity >98%). ¹H NMR (400MHz, DMSO-d₆), δ 9.57-9.71 (m), 8.75 (d), 8.42 (d), 8.15 (d), 7.14-7.29(m), 4.70-4.79 (m), 4.40-4.68 (m), 3.95-4.03 (m), 3.73-3.80 (m),3.37-3.61 (m), 2.97-3.31 (m), 2.79-2.88 (m), 2.66-2.76 (m), 2.54-2.58(m), 2.31-2.43 (m), 1.94-2.29 (m), 1.57-1.91 (m), 1.21-1.52 (m),0.85-1.10 (m), 0.74-0.82 (m)

Preparation of1,2-dimethyl-D-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#153)

Step 1 Synthesis of 1,2-dimethyl-D-proline (#152)

To a parr flask containing 2-methyl-D-proline (432 mg, 3.34 mmol, 1.0eq.), Formaldehyde 37 wt. % in water (1.0 mL, 37 mM, 11 eq.), 3.5 mL ofmethanol and 1 mL of water, Palladium 10 wt. % on Carbon (108 mg, 0.304mmol, 0.304 eq.) was added. The flask was placed on a parr shaker andallowed to shake under 30 psi of hydrogen for -48 hours. Hydrogen wasremoved and reaction was washed through a pad of celite, which wassubsequently washed with methanol. The organics where concentrated invacuo and then azeotroped with toluene affording #152 (517 mg, 100%) asa solid. ¹H NMR (400 MHz, methanol-d₄): δ [3.61-3.56 (m, 1H), 3.07-2.96(m, 1H), 2.68 (br s, 3H), 2.34-2.22 (m, 1H), 2.01-1.88 (m, 1H),1.87-1.73 (m, 1H), 1.40 (br s, 3H)].

Step 2 Synthesis of1,2-dimethyl-D-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(#153)

To a stirring mixture of #114 (240 mg, 0.379 mmol, 1.0 eq.), #152 (71mg, 0.49 mmol, 1.3 eq.), and HATU (188 mg, 0.49 mmol, 1.3 eq.) in 10 mLof dichloromethane, Hunig's base (0.27 mL, 4.1 mM, 4.1 eq.) was added.The reaction was allowed to stir at room temperature for 1 hour.Reaction was concentrated in vacuo. THF (6 mL) was added to crudematerial. To this stirring mixture LiOH (36 mg, 1.5 mmol, 4 eq.)dissolved in 2 mL of water was added. Reaction was allowed to stir atroom temperature for 1 hour. Reaction was concentrated in vacuo andresidue was purified by medium pressure reverse phase C18 chromatography(Gradient: 5% to 40% acetonitrile in water with 0.02% TFA in each phase)#153 (220 mg, 78%) as a white solid. LC-MS (Protocol Q): m/z 744.8[M+H⁺], retention time=1.16 minutes; HPLC (Protocol A at 45° C.): /z744.4 [M+H⁺], retention time=6.713 minutes (purity >98%). ¹H NMR (400MHz, DMSO-d₆), δ 9.72-9.85 (m), 8.65 (t), 8.41 (d), 8.14 (d), 7.14-7.28(m), 4.69-4.79 (m), 4.38-4.53 (m), 3.95-4.04 (m), 3.73-3.79 (m),3.37-3.62 (m), 3.13-3.33 (m), 2.95-3.10 (m), 2.79-2.89 (m), 2.67-2.75(m), 2.00-2.46 (m), 1.61-1.90 (m), 1.22-1.54 (m), 1.02-1.09 (m),0.95-1.01 (m), 0.85-0.94 (m), 0.75-0.83 (m)

Preparation ofN˜2˜-[2,2-dimethyl-3-(methylamino)propanoyl]-N-{(1S,2R)-2-methoxy-4-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#154)

Step 1 Synthesis ofN˜2˜-[2,2-dimethyl-3-(methylamino)propanoyl]-N-{(1S,2R)-2-methoxy-4-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#154)

To vial containing #50 (100 mg, 0.152 mmol, 1.0 eq.) and 1 mL ofdichloromethane, 2,2-dimethyl-3-(methylamino)propanoic acid (36 mg,0.152 mmol, 1.0 eq.) was added followed by Hunig's base (0.080 mL, 0.456mmol, 3.0 eq.) and HATU (66 mg, 0.17 mmol, 1.1 eq.). The reaction wasallowed to stir at room temperature for 1 hour. The reaction wasconcentrated in vacuo and then taken up in ethyl acetate before beingwashed two times with 1M HCl and once with brine. The organic layer wasdried over sodium sulfate and decanted. The reaction was concentrated invacuo. Dioxane (1 ml) was added followed by 4M HCl in dioxane (1.0 mL,4.0 mmol, 26 eq.). The reaction was allowed to stir at room temperaturefor ˜12 hours. The reaction was concentrated in vacuo. The crudematerial was purified by medium pressure reverse phase C18chromatography (Gradient: 10% to 100% acetonitrile in water with 0.02%TFA in each phase) yielding #154 (55.8 mg, 41%) as a solid. LC-MS(Protocol Q): m/z 771.8 [M+H⁺]. ¹H NMR (400 MHz, DMSO-d₆), δ 8.70 (d),8.45 (d), 7.90-8.15 (m), 7.82 (d), 7.75 (d), 7.55 (dd), 7.40 (dd),6.90-7.10 (m), 5.10-5.30 (m), 4.45-4.55 (b), 4.30-4.45 (m), 4.20-4.30(m), 3.75-3.90 (m), 3.50-3.60 (m), 3.15-3.40 (m), 3.05-3.15 (m),2.85-3.05 (m), 2.60-2.85 (m), 2.25-2.40 (m), 1.80-2.25 (m), 1.70-1.80(m), 1.20-1.60 (m), 0.80-1.10 (m), 0.05-0.80 (m).

Preparation of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[2,2-dimethyl-3-(methylamino)propanoyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#155)

Step 1 Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[2,2-dimethyl-3-(methylamino)propanoyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#155)

To vial containing #114 (96.2 mg, 0.152 mmol, 1.0 eq.) and 1 mL ofdichloromethane, 2,2-dimethyl-3-(methylamino)propanoic acid (36.1 mg,0.152 mmol, 1.0 eq.) was added followed by Hunig's base (0.080 mL, 0.456mmol, 3.0 eq.) and HATU (66 mg, 0.17 mmol, 1.1 eq.). The reaction wasallowed to stir at room temperature for 1 hour. The reaction wasconcentrated in vacuo and then taken up in ethyl acetate before beingwashed two times with 1M HCl and once with brine. The organic layer wasdried over sodium sulfate and decanted. The reaction was concentrated invacuo. Dioxane (1 ml) was added followed by 4M HCl in dioxane (1.0 mL,4.0 mmol, 26 eq.). The reaction was allowed to stir at room temperaturefor ˜12 hours. The reaction was concentrated in vacuo. The crudematerial was purified by medium pressure reverse phase C18chromatography (Gradient: 10% to 100% acetonitrile in water with 0.02%TFA in each phase) yielding #155 (22.2 mg, 17%). ¹H NMR (400 MHz,DMSO-d₆), δ 8.55 (d), 8.22 (d), 8.15-8.35 (m), 7.90-8.05 (m) 7.10-7.25(m) 4.70-4.80 (m), 4.55-4.65 (m), 4.45-4.52 (m), 3.93-4.00 (m),3.72-3.78 (m), 3.60-3.70 (m), 3.50-3.60 (m), 3.40-3.50 (m), 2.80-3.30(m), 2.45-2.60 (m), 2.00-2.45 (m), 1.60-1.80 (m), 1.35-1.50 (m),1.10-1.35 (m).

Preparation of methylN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2S)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#158) and methylN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2R)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#159)

Step 1 (Synthesis of(2S)-1-(tert-butoxycarbonyl)-2-methylpiperidine-2-carboxylic acid (#156)and (2R)-1-(tert-butoxycarbonyl)-2-methylpiperidine-2-carboxylic acid(#157)

1-(tert-butoxycarbonyl)-2-methylpiperidine-2-carboxylic acid (500 mg,2.06 mmol, 1 eq.) was separated by supercritical fluid chromatography(Column: Chiralcel OJ-H, 250×21 mm; Eluent: 90:10 carbondioxide/ethanol; Flow Rate: 65 g/min; to give the correspondingenantiomers. The first eluting peak (retention time=1.57 minutes) wasisolated to give #156 as a gum (140 mg, 28%) (stereochemistryarbitrarily assigned as the S enantiomer). ¹H NMR (400 MHz, CDCl₃) δ3.83-3.90 (m, 1H), 2.93-3.01 (m, 1H), 1.87-1.97 (m, 1H), 1.67-1.77 (m,3H), 1.48-1.66 (m, 2H), 1.46 (s, 3H), 1.44 (s, 9H). Optical rotation:[α]_(D)25−21.7° (c 0.40, chloroform). The second eluting peak (retentiontime=2.22 minutes) was isolated to give #157 as an oil (255 mg, 51%)(stereochemistry arbitrarily assigned as the R enantiomer). ¹H NMR (400MHz, CDCl₃) δ 3.83-3.90 (m, 1H), 2.93-3.01 (m, 1H), 1.87-1.97 (m, 1H),1.67-1.77 (m, 3H), 1.48-1.66 (m, 2H), 1.46 (s, 3H), 1.44 (s, 9H).Optical rotation: [α]_(D)25+30.2° (chloroform).

Step 2A Synthesis of methylN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2S)-2-methylpiperidin-2-yl]carbonyl}-Lvalyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#158)

To a solution of #156 (8.3 mg, 0.034 mmol, 1 eq.) in dichloromethane(0.3 mL) and N,N-dimethylformamide (0.05 mL), was addedN,N-diisopropylethylamine (0.018 mL, 0.102 mmol, 3 eq.), followed byHATU (16.1 mg, 0.041 mmol, 1.2 eq.). The reaction was stirred for 15minutes and #114 (23.4 mg, 0.037 mmol, 1.1 eq.) was added and stirred atroom temperature for 18 hours. The reaction was diluted withdichloromethane (2.5 mL) and 10% citric acid (1.5 mL) was added. Thelayers were separated using a phase separator cartridge and the aqueouslayer extracted with dichloromethane (2×2.5 mL) and the combined organiclayers were concentrated in vacuo. The residue was dissolved indichloromethane (4 mL) and trifluoroacetic acid (0.5 mL) was added. Thereaction was stirred at room temperature for 2 hours, then concentratedin vacuo. Purification by reverse phase chromatography (method M*)afforded #158 (10.6 mg, 49%). HPLC (Protocol T): m/z 758.4 [M+H⁺],retention time=2.53 minutes (purity >99%). ¹H NMR (400 MHz, DMSO-d₆), δ8.74-8.90 (m), 8.49-8.55 (m), 8.24 (d), 8.08-8.12 (m), 7.94-8.01 (m),7.14-7.26 (m), 4.71-4.77 (m), 4.57-4.68 (m), 4.44-4.55 (m), 3.94-4.0(m), 3.73-3.78 (m), 3.40-3.72 (m), 3.16-3.32 (m), 2.98-3.16 (m),2.82-2.92 (m), 2.47-2.56 (m), 2.38-2.44 (m), 2.20-2.37 (m), 2.08-2.19(m) 1.74-1.88 (m), 1.61-1.73 (m), 1.52-1.59 (m), 1.22-1.52 (m), 1.05(dd), 0.94-1.00 (m), 0.85-0.93 (m), 0.74-0.79 (m).

Step 2B Synthesis of methylN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2R)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#159)

To a solution of #157 (7.8 mg, 0.032 mmol, 1 eq.) in dichloromethane(0.3 mL) and N,N-dimethylformamide (0.05 mL), was added N,N-diisopropylethylamine (0.017 mL, 0.096 mmol, 3 eq.) followed by HATU(14.9 mg, 0.038 mmol, 1.2 eq.). The reaction was stirred for 15 minutesand #114 (22.1 mg, 0.035 mmol, 1.1 eq.) was added and stirred at roomtemperature for 3 hours and then concentrated in vacuo. Purification ofthe residue by silica gel chromatography (Gradient: 0 to 80% acetone inheptane) afforded a white solid which was dissolved in dioxane (0.2 mL)and 4N HCl in dioxane (0.2 mL) was added. The reaction was stirred atroom temperature for 2 hours and additional 4N HCl in dioxane (0.1 mL)was added. The reaction was stirred for 2 hours at room temperature,concentrated in vacuo. Purification by reverse phase chromatography(Method M*) afforded #159 (6.6 mg, 33%). HPLC (Protocol T): m/z 758.4[M+H⁺], retention time=2.46 minutes (purity=89%). ¹H NMR (400 MHz,DMSO-d₆), δ 8.86-8.95 (m), 8.75-8.84 (m), 8.48-8.54 (m), 8.33-8.45 (m),8.22-8.27 (m), 8.17-8.19 (m), 7.99-8.12 (m), 7.83-7.91 (m), 7.13-7.29(m), 7.04-7.08 (m), 4.69-4.76 (m), 4.55-4.66 (m), 4.45-4.53 (m),3.96-4.01 (m), 3.41-3.78 (m), 3.28-3.33 (m), 3.24-3.27 (m), 3.16-3.23(m), 3.11-3.15 (m), 3.02-3.10 (m), 2.93-3.02 (m), 2.91-2.93 (m),2.84-2.91 (m), 2.76-2.82 (m), 2.69-2.71 (m), 2.60-2.63 (m), 2.53-2.55(m), 2.47-2.53 (m), 2.40-2.46 (m), 2.30-2.38 (m), 2.20-2.30 (m),2.06-2.17 (m), 1.75-1.87 (m), 1.52-1.74 (m), 1.35-1.51 (m), 1.14-1.34(m), 1.01-1.08 (m), 0.92-1.0 (m), 0.85-0.94 (m), 0.74-0.82 (m).

Preparation ofN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2S)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt (#162) andN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2R)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt. (#163)

Step 1A Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(2S)-1-(tert-butoxycarbonyl)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate(#160)

To a solution of #156 (106 mg, 0.436 mmol, 1 eq.) in dichloromethane (3mL) and N,N-dimethylformamide (0.5 mL) was added diisopropylethylamine(0.228 mL, 1.31 mmol, 3 eq.) followed by HATU (205 mg, 0.523 mmol, 1.2eq.). The reaction was stirred for 15 minutes and #114 (276 mg, 0.436mmol, 1 eq.) was added and stirred at room temperature for 2 hours. Thereaction was diluted with dichloromethane (10 mL) and washed with 10%citric acid (3×5 mL). The organic layer was dried over sodium sulfate,filtered and the filtrate concentrated in vacuo. Purification of theresidue by silica gel chromatography (Gradient: 0 to 80% acetone inheptane) afforded #160 (145 mg, 39%). LC-MS (protocol Q1): m/z 858.8[M+H⁺], retention time=1.12 minutes.

Step 1B Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(2R)-1-(tert-butoxycarbonyl)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate(#161)

To a solution of #157 (109 mg, 0.448 mmol, 1 eq.) in dichloromethane (3mL) and N,N-dimethylformamide (0.5 mL), was added diisopropylethylamine(0.234 mL, 1.34 mmol, 3 eq.) followed by HATU (205 mg, 0.538 mmol, 1.2eq.). The reaction stirred for 15 minutes and #114 (284 mg, 0.448 mmol,1 eq.) was added. After stirring at room temperature for 2 hours, themixture was diluted with dichloromethane (10 mL), washed with 10% citricacid (3×5 mL). The organic layer was dried over sodium sulfate, filteredand concentrated in vacuo. Purification of the residue by silica gelchromatography (Gradient: 0 to 100% acetone in heptane) afforded #161(185 mg, 48%). LC-MS (Protocol Q): m/z 858.3 [M+H⁺], retention time=2.25minutes.

Step 2A Synthesis ofN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2S)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt (#162)

To a solution of #160 (145 mg, 0.169 mmol, 1 eq.) in tetrahydrofuran(1.25 mL) was added lithium hydroxide (8 mg, 0.338 mmol, 2 eq.)dissolved in water (0.75 mL). The reaction was stirred at roomtemperature for 2 hours and evaporated to dryness in vacuo. The residuewas dissolved in dichloromethane (2.5 mL) and trifluoroacetic acid (1mL) was added. The reaction was stirred for 30 minutes, concentrated invacuo and purified by medium pressure reverse phase C18 chromatography(Gradient: 0% to 100% acetonitrile in water with 0.02% TFA in eachphase) to afford the title compound #162 (145 mg, quantitative) as awhite solid. HPLC (Protocol U): m/z 744.5 [M+H+], retention time=7.121minutes (purity=98%). ¹H NMR (400 MHz, DMSO-d₆). δ 8.76-8.96 (m),8.52-8.58 (m), 8.38-8.43 (m), 8.11-8.16 (m), 7.27-7.30 (m), 7.12-7.27(m), 7.01-7.05 (m), 4.71-4.79 (m), 4.48-4.67 (m), 4.39-4.47 (m),3.79-4.22 (m), 3.71-3.78 (m), 3.38-3.57 (m), 3.22-3.30 (m), 3.14-3.23(m), 3.07-3.13 (m), 2.96-3.06 (m), 2.76-2.87 (m), 2.66-2.68 (m),2.47-2.57 (m), 2.42-2.44 (m), 2.06-2.40 (m), 1.73-1.89 (m), 1.51-1.72(m), 1.36-1.49 (m), 1.20-1.35 (m), 1.00-1.09 (m), 0.95-0.99 (m),0.83-0.94 (m), 0.73-0.80 (m).

Step 2B Synthesis ofN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(N-{[(2R)-2-methylpiperidin-2-yl]carbonyl}-L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt (#163)

Compound #161 (185 mg, 0.216 mmol, 1 eq.) was converted to the crudetitle compound #163, using the procedure described for the preparationof #162. The crude material was purified by medium pressure reversephase C18 chromatography (Gradient: 0% to 85% acetonitrile in water with0.02% TFA in each phase) to yield #163 (127 mg, 68%) as a white solid.HPLC (Protocol U): m/z 744.5 [M+H+], retention time=7.077 minutes(purity=98%). ¹H NMR (400 MHz, DMSO-d₆). δ 8.79-8.99 (m), 8.36-8.49 (m),8.12-8.17 (m), 7.31-7.34 (m), 7.11-7.27 (m), 7.05-7.09 (m), 4.71-4.77(m), 4.54-4.68 (m), 4.40-4.53 (m), 3.88-4.39 (m), 3.71-3.77 (m),3.39-3.58 (m), 3.22-3.32 (m), 3.10-3.22 (m), 3.04-3.09 (m), 2.92-3.03(m), 2.77-2.88 (m), 2.68-2.71 (m), 2.47-2.57 (m), 2.43-2.45 (m),2.30-2.42 (m), 2.03-2.29 (m), 1.74-1.88 (m), 1.52-1.73 (m), 1.37-1.51(m), 1.17-1.37 (m), 1.00-1.07 (m), 0.95-0.99 (m), 0.84-0.93 (m),0.73-0.81 (m).

Preparation of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#172) and methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#173)

Step 1 Synthesis of methyl(3R)-1-benzyl-3-fluoropyrrolidine-3-carboxylate (#164) and methyl(3S)-1-benzyl-3-fluoropyrrolidine-3-carboxylate (#165)

Known (methyl 1-benzyl-3-fluoropyrrolidine-3-carboxylate (3900 mg, 16.4mmol, 1 eq.) was separated by supercritical fluid chromatography(Column: Chiralpak IC, 250×21 mm; Eluent: 95:5 carbon dioxide/propanol;Flow Rate: 65 g/min; to give the corresponding enantiomers. The firsteluting peak (retention time=3.37 minutes) was isolated to afford #164(1720 mg, 36%) as a single enantiomer (stereochemistry arbitrarilyassigned as R enantiomer). ¹H NMR (400 MHz, TMS-CDCl₃; δ 7.17-7.30 (m,5H), 3.74 (s, 3H), 3.65 (d, J=12.9 Hz, 1H), 3.63 (d, J=12.9 Hz, 1H),2.86-3.03 (m, 3H), 2.61 (q, J=8.0 Hz, 1H), 2.34-2.46 (m, 1H), 2.13-2.26(m, 1H). Optical rotation: [α]_(D)25+24.7° (chloroform). The secondeluting peak (retention time=3.91 minutes) was isolated to afford #165(1600 mg, 33%) as a single enantiomer (stereochemistry arbitrarilyassigned as S enantiomer). ¹H NMR (400 MHz, CDCl₃; (CH₃)₄Si), δ7.17-7.30 (m, 5H), 3.74 (s, 3H), 3.65 (d, J=12.9 Hz, 1H), 3.63 (d,J=12.9 Hz, 1H), 2.86-3.03 (m, 3H), 2.61 (q, J=8.0 Hz, 1H), 2.34-2.46 (m,1H), 2.13-2.26 (m, 1H). Optical rotation: [α]_(D)25-23.3° (chloroform).

Step 2A Synthesis of 1-tert-butyl 3-methyl(3R)-3-fluoropyrrolidine-1,3-dicarboxylate (#166)

To a solution containing #164 (355 mg, 1.50 mmol, 1 eq.) andDi-tert-butyl carbonate (400 mg, 1.8 mmol, 1.2 eq.) in methanol (15.5mL) was added 10% Pd/C (70 mg). The reaction was hydrogenated at 45 psiin a Parr shaker at for 22 hours, filtered over celite, and the filtrateconcentrated in vacuo and purified by silica gel chromatography(Gradient: 0 to 30% ethyl acetate in heptane) to afford #166 as a clearoil. (272 mg, 74%). ¹H NMR (400 MHz, CDCl₃), δ 3.87 (s, 3H), 3.85-3.66(m, 3H), 3.56 (m, 1H), 2.53-2.28 (m, 2H), 1.51 (s, 9H).

Step 2B Synthesis of 1-tert-butyl 3-methyl(3S)-3-fluoropyrrolidine-1,3-dicarboxylate (#167)

Compound #165 (362 mg, 1.53 mmol, 1 eq.) was converted to #167 in 63%yield using the method described above for #164. ¹H NMR (400 MHz,CDCl₃), δ 3.87 (s, 3H), 3.85-3.66 (m, 3H), 3.56 (m, 1H), 2.53-2.28 (m,2H), 1.51 (s, 9H).

Step 3A Synthesis of(3R)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidine-3-carboxylic acid(#168)

To a solution of #166 (272 mg, 1.10 mmol, 1 eq.) dissolved in methanol(2.96 mL) was added an aqueous solution of sodium hydroxide (2.5 M, 0.88mL) and the reaction was stirred at room temperature for 3.5 hours. Thereaction was quenched with 10% aqueous citric acid (5 mL), ethyl acetate(100 mL) was added, and the layers separated. The organic layer waswashed with 10% citric acid, water, and brine, dried over sodiumsulfate, filtered and concentrated in vacuo to afford #168 as a whitesolid. (253 mg, 99%). ¹H NMR (400 MHz, CDCl₃), δ 3.96-3.69 (m, 3H), 3.59(m, 1H), 2.59-2.33 (m, 2H), 1.51 (s, 9H). LC-MS (Protocol Q1): m/z 232.1[M−H+], retention time=0.67 minutes. Chiral HPLC retention time: 3.39min (purity=99%). (Column: Chiralpak AD-H, 4.6 mm×25 cm, mobile phase5-60% CO₂/Methanol, flow rate 3.0 mL/min); Optical rotation: [α]_(D)254.8 (c=0.52, MeOH)

Step 3B Synthesis of(3S)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidine-3-carboxylic acid(#169)

To a solution of #167 (238 mg, 0.963 mmol, 1 eq.) dissolved in methanol(2.6 mL) was added an aqueous solution of sodium hydroxide (2.5 M, 0.88mL) and the reaction was stirred at room temperature for 3 hours. Thereaction was then quenched with 10% aqueous citric acid (5 mL) and ethylacetate (100 mL) was added, and the layers were separated. The organiclayer was washed with 10% citric acid, water, and brine, then dried oversodium sulfate, filtered and concentrated in vacuo to afford #169 as awhite solid (221 mg, 99%). ¹H NMR (400 MHz, CDCl₃), δ 3.96-3.69 (m, 3H),3.59 (m, 1H), 2.59-2.33 (m, 2H), 1.51 (s, 9H). LC-MS (Protocol Q1): m/z232.1 [M−H+], retention time=0.67 minutes. Chiral HPLC retention time:3.95 min (purity=98%)(Column: Chiralpak AD-H, 4.6 mm×25 cm, mobile phase5-60% CO₂/Methanol, flow rate 3.0 mL/min); Optical rotation:[α]_(D)25−3.6 (c=0.55, MeOH)

Step 4A Synthesis of lithium(3R)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidine-3-carboxylate (#170)

To a solution of #168 (50 mg, 0.21 mmol, 1 eq.) in methanol (0.2 mL) wasadded a solution of lithium hydroxide (9.2 mg, 0.38 mmol, 1.8 eq.)dissolved in water (0.1 mL). Next, tetrahydrofuran (0.3 mL) was addedand the reaction was stirred at 45° C. for 18 hours. The reaction wasconcentrated in vacuo and the material was azeotroped (3×) with toluene(2 mL) to obtain #170 (51 mg, 100%) as a white solid which was used inthe next step without further purification.

Step 4B Synthesis of lithium(3S)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidine-3-carboxylate (#171)

To a solution of #169 (75 mg, 0.32 mmol, 1 eq.) in methanol (0.3 mL) wasadded a solution of lithium hydroxide (13.8 mg, 0.572 mmol, 1.8 eq.) inwater (0.4 mL). Next, tetrahydrofuran (0.45 mL) was added and thereaction was stirred at 45° C. for 18 hours. The reaction wasconcentrated in vacuo and the material was azeotroped (3×) with toluene(4 mL) to obtain #171 (77 mg, 100%) as a white solid, which was used inthe next step without further purification.

Step 5A Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#172)

To a suspension of #168 (36.9 mg, 0.158 mmol, 1 eq.) and #114 (100 mg,0.158 mmol, 1.0 eq.) in N,N-dimethylformamide (0.8 mL) anddichloromethane (3.6 mL) was added N,N-diisopropylethylamine (0.083 mL,0.474 mmol, 3 eq.) followed by HATU (60.7 mg, 0.158 mmol, 1.0 eq.) andthe reaction was stirred at room temperature for 18 hours. The reactionwas diluted with ethyl acetate and was washed successively with water,10% aqueous citric acid (WN), and brine. The organic layer was driedover sodium sulfate and concentrated in vacuo to give 220 mg (164% oftheory) of a crude intermediate. A portion of this crude intermediate(50 mg, 23%) was dissolved in dichloromethane (1.5 mL) andtrifluoroacetic acid (0.4 mL) was added. The mixture was stirred at roomtemperature for 2 hours and evaporated to dryness in vacuo. Purificationby reverse phase chromatography (Method M*) afforded #172 (15.8 mg, 51%)LC-MS (Protocol Q): m/z 748.9 [M+H⁺] retention time=1.29 minutes. ¹H NMR(DMSO-d₆), δ 9.16-9.43 (m), 8.48-8.53 (m), 8.40-8.44 (m), 8.34-8.39 (m),8.22-8.30 (m), 8.09-8.16 (m), 7.87-7.91 (m), 7.77-7.83 (m), 7.12-7.24(m), 4.56-4.72 (m), 4.41-4.54 (m), 3.92-4.00 (m), 3.70-3.75 (m),3.39-3.66 (m), 3.20-3.25 (m), 3.12-3.20 (m), 2.97-3.11 (m), 2.94 (br s),2.75-2.89 (m), 2.63-2.69 (m), 2.47-2.54 (m), 2.29-2.45 (m), 2.15-2.27(m), 2.02-2.15 (m), 1.57-1.87 (m), 1.33-1.51 (m), 1.19-1.30 (m),1.02(dd), 0.82-0.97 (m), 0.70-0.79 (m).

Step 5B Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate,trifluoroacetic acid salt (#173)

To a suspension of #169 (36.9 mg, 0.158 mmol, 1 eq.) and #114 (100.0 mg,0.158 mmol, 1 eq.) in N,N-dimethylformamide (0.8 mL) and dichloromethane(3.6 mL) was added N,N-diisopropylethylamine (0.083 mL, 0.474 mmol, 3eq.) was added, followed by HATU (60.7 mg, 0.158 mmol, 1 eq.). Thereaction was stirred at room temperature for 14 hours, diluted withethyl acetate, and washed successively with water, 10% aqueous citricacid (W/V), and brine. The organic layer was dried over sodium sulfateand concentrated in vacuo to give 180 mg (134% of theory) of a crudeintermediate. A portion of this crude intermediate (50 mg, 27%) wasdissolved in dichloromethane (1.5 mL) and trifluoroacetic acid (0.4 mL)was added and the mixture stirred at room temperature for 2 hours andevaporated to dryness in vacuo. Purification by reverse phasechromatography (Method M*) afforded #173 (17.6 mg, 45%). LC-MS (ProtocolQ): m/z 748.9 [M+H+] retention time=1.29 minutes. ¹H NMR (DMSO-d₆) δ9.35-9.50 (m), 9.22-9.34 (m), 8.47-8.52 (m), 8.39-8.45 (m), 8.30-8.37(m), 8.22-8.24 (m), 8.09-8.13 (m), 7.80-7.85 (m), 7.67-7.72 (m),7.09-7.24 (m), 6.97-6.98 (m), 4.65-4.72 (m), 4.55-4.64 (m), 4.41-4.50(m), 3.92-3.99 (m), 3.42-3.75 (m), 3.32-3.39 (m), 3.25-3.31 (m),3.21-3.24 (m), 3.11-3.20 (m), 3.06-3.11 (M), 2.97-3.05 (m), 2.95 (br s),2.83-2.88 (m), 2.76-2.82 (m), 2.65-2.70 (m), 2.44-2.54 (m), 2.15-2.42(m), 2.02-2.14 (m), 1.57-1.84 (m), 1.33-1.48 (m), 1.19-1.30 (m), 1.02(dd), 0.92-0.97 (m), 0.82-0.91 (m), 0.70-0.77 (m).

Preparation of(2S)—N-[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide,hydrochloride salt (#178) and(2R)—N-[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide,hydrochloride salt (#180)

Step 1 Synthesis of pentafluorophenyl(3R,4S,5S)-4-[{N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoate(#174)

To a solution of #@5 (19.43 g, 37.03 mmol, 1 eq.) in dichloromethane(100 mL) and pyridine (5.86 g, 74.1 mmol, 2 eq.) was addedpentafluorophenyl trifluoroacetate (20.7 g, 74.1 mmol, 2 eq.) and thereaction was stirred at room temperature for 1 hour. The reaction wasconcentrated in vacuo and purified by silica gel chromatography(Gradient: 0 to 52% ethyl acetate in heptane) to afford #174 (23.58 g,92%) as a yellow oil. LC-MS (Protocol Q1): m/z 691.2 [M+H+], retentiontime=1.23 minutes.

Step 2 Synthesis ofN-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N˜2˜-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valinamide(#175)

To a solution of #174 (706 mg, 1.02 mmol, 1 eq.) and #64 (311 mg, 1.02mmol, 1 eq.) in dichloromethane (3 mL) was addedN,N-diisopropylethylamine (400 mg, 3.07 mmol, 3 eq.). After 18 hours ofstirring at room temperature, the reaction was concentrated in vacuo andpurified by silica gel chromatography (Gradient: 0 to 100% ethyl acetatein heptane) to afford #175 (560 mg, 68%) as a white solid. LC-MS(Protocol Q1): m/z 611.8 [M+H+], retention time=1.15 minutes.

Step 3 Synthesis ofN-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#176)

According to the general procedure A, from #175 (560 mg, 0.690 mmol, 1eq.) in dichloromethane (9 mL), and N,N-diethylamine (6.0 mL), wassynthesized the crude desired compound, which was purified by by silicagel chromatography (Gradient: 0 to 50% methanol in dichloromethane) toafford #176 (351 mg, 87%) as a yellow oil. LC-MS (Protocol Q1): m/z589.5 [M+H⁺], retention time=0.72 minutes.

Step 4A Synthesis of tert-butyl(2S)-2-{[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpiperidine-1-carboxylate(#177)

According to the general procedure D, from #176 (100 mg, 0.170 mmol, 1eq.), #156 (53.8 mg, 0.221 mmol, 1.3 eq.), dichloromethane (4.5 mL),HATU (84.9 mg, 0.221 mmol, 1.3 eq.) and N,N-diisopropylethylamine (0.123mL, 0.697 mmol, 4.1 eq.), was synthesized the crude desired material,which was purified by silica gel chromatography (Gradient: 0 to 100%ethyl acetate in heptane) to afford #177 (145 mg, assume quantitativeyield) as a white solid. LC-MS (Protocol Q1): m/z 814.7 [M+H⁺],retention time=1.14 minutes.

Step 4B Synthesis of tert-butyl(2R)-2-{[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpiperidine-1-carboxylate(#179)

According to the general procedure D, from #176 (100 mg, 0.170 mmol, 1eq.), #157 (53.8 mg, 0.221 mmol, 1.3 eq.), dichloromethane (4.5 mL),HATU (84.9 mg, 0.221 mmol, 1.3 eq.) and N,N-diisopropylethylamine (0.123mL, 0.697 mmol, 4.1 eq.), was synthesized the crude desired material,which was purified by silica gel chromatography (Gradient: 0 to 100%ethyl acetate in heptane) to afford #179 (155 mg, assume quantitativeyield) as a white solid. LC-MS (Protocol Q1): m/z 814.7 [M+H⁺],retention time=1.14 minutes.

Step 5A Synthesis of(2S)-N-[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide, hydrochloride salt (#178)

According to the general procedure C, from #177 (143 mg, 0.176 mmol, 1eq.) and 4M solution of hydrochloric acid in dioxane (2.0 mL) wassynthesized the desired material as a gum (145 mg). A portion of thiscrude residue (25 mg) was azeotroped with a mixture ofmethanol/acetonitrile to afford #178 (20 mg, 89% yield) as a whitesolid. ¹H NMR (400 MHz, DMSO-d₆), δ 8.96-9.07 (m), 8.79-8.96 (m), 8.58(d), 8.02-8.08 (m), 7.77-7.83 (m), 7.25-7.31 (m), 7.19-7.24 (m),6.56-6.67 (m), 6.12-6.21 (m), 5.13-5.22 (m), 4.72-4.81 (m), 4.63-4.70(m), 4.50-4.59 (m), 4.07-4.16 (m), 3.98-4.05 (m), 3.80-3.86 (m),3.55-3.76 (m), 3.46-3.54 (m), 3.38-3.44 (m), 3.26-3.35 (m), 3.18-3.24(m), 3.05-3.18 (m), 2.98-3.04 (m), 2.40-2.55 (m), 2.27-2.35 (m),2.08-2.27 (m), 1.74-1.98 (m), 1.50-1.74 (m), 1.20-1.46 (m), 0.73-1.16(m). LC-MS (Protocol Q1): m/z 714.6 [M+H⁺], retention time=0.76 minutes.HPLC (Protocol U): m/z 714.5 [M+H⁺] retention time=7.124 minutes(purity=91%).

Step 5B Synthesis of(2R)—N-[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide,hydrochloride salt (#180)

According to the general procedure C, from #179 (162 mg, 0.199 mmol, 1eq.) and 4M solution of hydrochloric acid in dioxane (2.0 mL) wassynthesized the desired material as a gum (155 mg). A portion of thisgum (25 mg) was azeotroped with a 1/1 mixture of methanol/acetonitrileto afford #180 (20 mg, 83%) as a solid. ¹H NMR (400 MHz, DMSO-d₆), δ9.02-9.13 (m), 8.83-8.93 (m), 8.39-8.46 (m), 8.00-8.06 (m), 7.78 (t),7.24-7.30 (m), 7.16-7.21 (m), 6.54-6.65 (m), 6.09-6.19 (m), 5.11-5.18(m), 4.69-4.78 (m), 4.59-4.68 (m), 4.46-4.56 (m), 4.08-4.13 (m),3.95-4.03 (m), 3.77-3.85 (m), 3.54-3.73 (m), 3.43-3.53 (m), 3.37-3.42(m), 3.24-3.33 (m), 3.16-3.22 (m), 3.03-3.15 (m), 2.99-3.02 (m),2.89-2.98 (m), 2.65-2.76 (m), 2.41-2.54 (m), 2.15-2.39 (m), 2.07-2.15(m), 1.51-1.94 (m), 1.49 (d), 1.38 (t), 1.20-1.32 (m), 1.02-1.09 (m),0.84-0.97 (m), 0.73-0.81 (m). LC-MS (Protocol Q1): m/z 714.6 [M+H⁺],retention time=0.76 HPLC (Protocol U): m/z 714.4 [M+H⁺], retentiontime=7.409 minutes (purity=90%).

Preparation of2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,formic acid salt (#182) andN-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-˜-2˜-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#184) andN-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N˜2˜-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#186)

Step 1A Synthesis of1-(tert-butoxycarbonyl)-2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#181)

According to the general procedure D, from #176 (100 mg, 0.170 mmol, 1eq.), (S)-1-(tert-butoxycarbonyl)-2-methyl-L-proline (50.7 mg, 0.221mmol, 1.3 eq.), dichloromethane (4.3 mL), HATU (84.9 mg, 0.221 mmol, 1.3eq.) and N,N-diisopropylethylamine (0.123 mL, 0.697 mmol, 4.1 eq.), wassynthesized the crude desired material, which was purified by silica gelchromatography (Gradient: 0 to 100% ethyl acetate in heptane) to afford#181 (142 mg, assume quantitative yield). LC-MS (Protocol Q1): m/z 800.6[M+H⁺], retention time=1.11 minutes.

Step 1B Synthesis of tert-butyl(3R)-3-{[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-3-fluoropyrrolidine-1-carboxylate(#183)

To a solution of #170 (18.2 mg, 0.076 mmol, 1 eq.) in dichloromethane(1.8 mL) and N,N-dimethylformamide (0.3 mL) was addedN,N-diisopropylethylamine (0.040 mL, 0.228 mmol, 3 eq.) followed by HATU(29.2 mg, 0.076 mmol, 1 eq.). After stirring for 10 minutes at roomtemperature, #176 (45 mg, 0.076 mmol, 1 eq.) was added. The reaction wasstirred at room temperature for 18 hours and additional HATU (29 mg,0.076 mmol, 1 eq.) was added. After 8 hours the reaction wasconcentrated in vacuo to provide #183 (61.0 mg, quantitative) which wastaken into the next step without further purification. LC-MS (ProtocolQ1): m/z 826.6 [M+Na⁺], retention time=1.05 minutes.

Step 1C Synthesis of tert-butyl(3S)-3-{[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-3-fluorocyclopentanecarboxylate(#185)

To a solution of #171 (24 mg, 0.1 mmol, 1 eq.) in dichloromethane (2.35mL) and N,N-dimethylformamide (0.33 mL) was addedN,N-diisopropylethylamine (0.053 mL, 0.300 mmol, 3 eq.) followed by HATU(38.4 mg, 0.100 mmol, 1 eq.). After stirring at room temperature for 10minutes, #176 (58.9 mg, 0.1 mmol, 1 eq.) was added. The reaction wasstirred at room temperature for 18 hours and additional quantity of HATU(38.4 mg, 0.100 mmol, 1 eq.) and N,N-dimethylformamide (0.2 mL) wasadded and stirred for an additional 9 hours. The reaction wasconcentrated in vacuo to give #185 (80 mg, quantitative), which wastaken into the next step without further purification. LC-MS (ProtocolQ1): m/z 804.6 [M+H⁺], retention time=1.05 minutes.

Step 2A Synthesis of2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,formic acid salt (#182)

According to the general procedure C, from #181 (136 mg, 0.170 mmol, 1eq.) and 4M solution of hydrochloric acid in dioxane (2 mL) wassynthesized the desired material as a gum (142 mg). A portion of thiscrude residue (20 mg, 14%) was azeotroped with a 1/1 mixture ofmethanol/acetonitrile and then purified by reverse phase chromatography(Method O) to obtain #182 (10 mg, 57% over two steps) as a solid. HPLC(Protocol U): m/z 700.4 [M+H⁺], retention time=7.106 minutes(purity >90%). ¹H NMR (400 MHz, DMSO-d₆), δ 8.25-8.39 (m), 8.20-8.25(m), 7.96-7.99 (m), 7.74-7.77 (m), 6.55-6.63 (m), 6.10-6.18 (m),5.11-5.18 (m), 4.66-4.72 (m), 4.51-4.61 (m), 4.46-4.50 (m), 3.96-4.01(m), 3.37-3.86 (m), 3.20-3.36 (m), 3.11-3.19 (m), 3.03-3.11 (m),2.98-3.03 (m), 2.90-2.96 (m), 2.77-2.79 (m), 2.65-2.73 (m), 2.57-2.63(m), 2.47-2.56 (m), 2.36-2.46 (m), 2.26-2.32 (m), 2.14-2.25 (m),2.03-2.10 (m), 1.92-2.03 (m), 1.72-1.92 (m), 1.64-1.72 (m), 1.60-1.64(m), 1.50-1.59 (m), 1.39-1.48 (m), 1.23-1.32 (m), 1.18-1.22 (m),1.12-1.14 (m), 1.01-1.09 (m), 0.83-1.00 (m), 0.74-0.83 (m), 0.70-0.74(m).

Step 2B Synthesis ofN-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N˜2˜-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-N-methyl-L-valinamidetrifluoroacetic acid salt (#184)

According to the general procedure C, from #183 (61.1 mg, 0.076 mmol, 1eq.), dichloromethane (0.3 mL), and a 4M solution of hydrochloric acidin dioxane (0.9 mL) was synthesized the crude desired material (140 mg).A portion of the crude material (98 mg, 69%) was purified by reversephase chromatography (Method M*) to give #184 (7.6 mg, 20% over twosteps). HPLC (Protocol T): m/z 704.5 [M+H+], retention time=2.50 minutes(purity=84%). ¹H NMR (400 MHz, DMSO-d₆), δ 8.67-8.71 (m), 8.44-8.48 (m),8.27-8.33 (m), 8.22-8.27 (m), 7.97-8.03 (m), 7.84-7.89 (m), 7.74-7.81(m), 7.43-7.48 (m), 7.23-7.29 (m), 7.17-7.21 (m), 6.55-6.66 (m),6.10-6.19 (m), 5.10-5.19 (m), 4.66-4.75 (m), 4.51-4.63 (m), 3.96-4.04(m), 3.78-3.85 (m), 3.65-3.73 (m), 3.46-3.62 (m), 3.37-3.45 (m),3.24-3.37 (m), 3.21-3.24 (m), 3.13-3.21 (m), 3.02-3.13 (m), 2.95-3.00(m), 2.80-2.82 (m), 2.66-2.71 (m), 2.47-2.57 (m), 2.24-2.46 (m),2.09-2.24 (m), 1.95-2.05 (m), 1.49-1.93 (m), 1.22-1.34 (m), 1.02-1.09(m), 0.83-1.01 (m), 0.74-0.82 (m).

Step 2C Synthesis ofN-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N˜2˜-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-N-methyl-L-valinamide,trifluoroacetic acid salt (#186)

According to the general procedure C, from #185 (80.3 mg, 0.1 mmol, 1eq.), dichloromethane (0.4 mL), and a 4M solution of hydrochloric acidin dioxane (1.2 mL) was synthesized the crude desired material, in whicha portion (94 mg, 53%) was purified by reverse phase chromatography(Method M*) to give #186 (11.8 mg, 32% over two steps). ¹H NMR (400 MHz,DMSO-d₆), δ 8.85-9.07 (m), 8.29-8.41 (m), 7.99-8.04 (m), 7.76-7.84 (m),6.56-6.68 (m), 6.12-6.21 (m), 5.12-5.21 (m), 4.87-4.99 (m), 4.69-4.79(m), 4.49-4.67 (m), 3.98-4.06 (m), 3.80-3.87 (m), 3.64-3.76 (m),3.55-3.64 (m), 3.47-3.54 (m), 3.39-3.46 (m), 3.26-3.39 (m), 3.22-3.25(m), 3.18-3.22 (m), 3.06-3.14 (m), 2.98-3.01 (m), 2.55-2.57 (m),2.42-2.49 (m), 2.11-2.38 (m), 2.09 (s), 1.78-1.97 (m), 1.72-1.77 (m),1.51-1.71 (m), 1.24-1.36 (m), 1.07 (dd), 0.83-1.03 (m), 0.75-0.82 (m).HPLC (Protocol T): m/z 704.5 [M+H⁺], retention time=2.48 minutes(purity=100%).

Preparation of(2S)—N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide,formate salt (#188) and(2R)—N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yfl(methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide,formate salt (#190) andN˜2˜-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt. (#192) andN˜2˜-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt. (#194)

Step 1A Synthesis of tert-butyl(2S)-2-{[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpiperidine-1-carboxylate(#187)

According to the general procedure D, from #86 (280 mg, 0.4 mmol, 1eq.), #156 (100 mg, 0.4 mmol, 1 eq.), dichloromethane (5 mL), HATU (182mg, 0.48 mmol, 1.2 eq.) and N,N-diisopropylethylamine (100 mg, 0.8 mmol,2 eq.) was synthesized the crude desired material, which was purified bysilica gel chromatography (Gradient: 0.01 to 0.05% methanol indichloromethane) to afford #187 (220 mg, 62%) as a white solid. HPLC(Protocol V): m/z 883.57 [M+H⁺], retention time=3.23 minutes(purity=95%).

Step 1B Synthesis of tert-butyl(2R)-2-{[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpiperidine-1-carboxylate(#189)

According to the general procedure D, from #86 (400 mg, 0.6 mmol, 1eq.), #157 (146 mg, 0.6 mmol, 1 eq.), dichloromethane (10 mL), HATU (259mg, 0.72 mmol, 1.2 eq.) and N,N-diisopropylethylamine (158 mg, 1.2 mmol,2 eq.) was synthesized the crude desired material, which was purified bysilica gel chromatography (Gradient: 0.01 to 0.05% methanol indichloromethane) to afford #189 (220 mg, 37%) as a white solid. HPLC(Protocol W): m/z 883.7 [M+H⁺], retention time=4.12 minutes(purity=95%).

Step 1C Synthesis of tert-butyl(3R)-3-fluoro-3-{[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carboxylate(#191)

According to the general procedure D, from #86 (300 mg, 0.45 mmol, 1eq.), #168 (106 mg, 0.45 mmol, 1 eq.), dichloromethane (10 mL), HATU(194 mg, 0.54 mmol, 1.2 eq.) and diisopropylethylamine (117 mg, 0.9mmol, 2 eq.) was synthesized the crude desired material, which waspurified by reverse phase chromatography (Method P) to afford #191 (159mg, 40%) as a white solid. HPLC (Protocol X): m/z 873.4 [M+H⁺],retention time=3.32 minutes (purity=99%).

Step 1D Synthesis of tert-butyl(3S)-3-fluoro-3-{[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carboxylate(#193)

According to the general procedure D, from #86 (300 mg, 0.45 mmol, 1eq.), #169 (106 mg, 0.45 mmol, 1 eq.), dichloromethane (10 mL), HATU(194 mg, 0.54 mmol, 1.2 eq.) and diisopropylethylamine (117 mg, 0.9mmol, 2 eq.) was synthesized the crude desired material, which waspurified by reverse phase chromatography (Method P) to afford #193 (149mg, 37%) as a white solid. HPLC (Protocol X): m/z 873.4 [M+H⁺],retention time=3.34 minutes (purity=98%).

Step 2A Synthesis of(2S)—N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide,formic acid salt (#188)

According to the general procedure C, from #187 (20 mg, 0.023 mmol, 1eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution ofhydrochloric acid in dioxane (0.26 mL) was synthesized the crude desiredmaterial, which was purified by reverse phase chromatography (Method N*)to obtain #188 (11.6 mg, 61%,); HPLC (Protocol T): m/z 783.8 [M+H⁺],retention time=2.53 minutes (purity=96%). ¹H NMR (400 MHz, DMSO-d₆), δ8.82-8.87 (m), 8.60-8.63 (m), 8.26-8.29 (m), 7.84-7.90 (m), 7.75-7.81(m), 7.60-7.67 (m), 7.21-7.31 (m), 7.14-7.19 (m), 5.49-5.55 (m),5.37-5.42 (m), 4.69-4.75 (m), 4.59-4.65 (m), 4.50-4.56 (m), 3.95-4.01(m), 3.77-3.82 (m), 3.54-3.61 (m), 3.47-3.53 (m), 3.24-3.45 (m),3.14-3.23 (m), 3.03-3.08 (m), 2.96-3.03 (m), 2.78-2.80 (m), 2.64-2.73(m), 2.60-2.62 (m), 2.47-2.56 (m), 2.31-2.45 (m), 2.13-2.28 (m),2.00-2.07 (m), 1.92-1.99 (m), 1.71-1.86 (m), 1.58-1.70 (m), 1.50-1.56(m), 1.38-1.49 (m), 1.15-1.36 (m), 1.08-1.14 (m), 1.03-1.07 (m),0.90-1.02 (m), 0.83-0.90 (m), 0.73-0.80 (m), 0.70-0.73 (m).

Step 2B Synthesis of(2R)—N-[(2S)-1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]-2-methylpiperidine-2-carboxamide,formic acid salt. (#190)

According to the general procedure C, from #189 (20 mg, 0.022 mmol, 1eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution ofhydrochloric acid in dioxane (0.26 mL) was synthesized the crude desiredmaterial, which was purified by reverse phase chromatography (Method N*)to obtain #190 (13.2 mg, 73%) HPLC (Protocol T): m/z 783.7 [M+H⁺],retention time=2.5 minutes (purity=100%). ¹H NMR (400 MHz, DMSO-d₆), δ8.83-8.86 (m), 8.60-8.62 (m), 8.24-8.27 (m), 7.83-7.89 (m), 7.78-7.80(m), 7.75-7.77 (m), 7.64-7.66 (m), 7.60-7.63 (m), 7.20-7.31 (m),7.13-7.19 (m), 5.49-5.55 (m), 5.36-5.42 (m), 4.65-4.74 (m), 4.60-4.65(m), 4.50-4.56 (m), 3.95-4.01 (m), 3.76-3.81 (m), 3.53-3.62 (m),3.47-3.52 (m), 3.22-3.45 (m), 3.14-3.21 (m), 2.97-3.05 (m), 2.93-2.96(m), 2.79-2.86 (m), 2.76-2.78 (m), 2.65-2.68 (m), 2.48-2.56 (m),2.37-2.43 (m), 2.29-2.35 (m), 2.17-2.27 (m), 2.04-2.11 (m), 1.93-2.00(m), 1.70-1.85 (m), 1.53-1.69 (m), 1.36-1.48 (m), 1.28-1.36 (m),1.13-1.26 (m), 1.08-1.12 (m), 0.98-1.07 (m), 0.91-0.97 (m), 0.84-0.91(m), 0.73-0.78 (m).

Step 2C Synthesis ofN˜2˜-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt. (#192)

According to the general procedure C, from #191 (10 mg, 0.011 mmol, 1eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution ofhydrochloric acid in dioxane (0.13 mL) was synthesized the crude desiredmaterial, which was purified by reverse phase chromatography (Method M*)to obtain #192 (5.1 mg, 60%) HPLC (Protocol T): m/z 773.5 [M+H⁺],retention time=2.43 minutes (purity=100%). ¹H NMR (400 MHz, DMSO-d₆) δ9.32-9.44 (m), 9.17-9.21 (m), 8.91 (d), 8.63-8.69 (m), 8.38-8.43 (m),8.22-8.27 (m), 7.80 (dd), 7.66 (dd), 7.14-7.33 (m), 5.49-5.57 (m),5.37-5.45 (m), 4.10-4.78 (m), 3.97-4.06 (m), 3.77-3.83 (m), 3.33-3.65(m), 3.15-3.29 (m), 2.95-3.09 (m), 2.82-2.83 (m), 2.67-2.71 (m),2.55-2.57 (m), 2.32-2.54 (m), 2.10-2.31 (m), 2.09 (s), 1.72-1.90 (m),1.57-1.72 (m). 1.38-1.50 (m), 1.15-1.38 (m), 1.09 (dd), 0.85-1.0 (m),0.75-0.83 (m).

Step 2D Synthesis ofN˜2˜-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, trifluoroacetic acid salt. (#194)

According to the general procedure C, from #193 (10 mg, 0.011 mmol, 1eq.), dichloromethane (0.1 mL), acetonitrile (0.1 mL) and 4M solution ofhydrochloric acid in dioxane (0.13 mL) was synthesized the crude desiredmaterial, which was purified by reverse phase chromatography (Method M*)to obtain #194 (9 mg, 93%) HPLC (Protocol T): m/z 773.8 [M+H⁺],retention time=2.42 minutes (purity=100%). ¹H NMR (400 MHz, DMSO-d₆) δ9.39-9.52 (m), 9.21-9.35 (m), 8.90 (d), 8.63-8.69 (m), 8.42-8.48 (m),8.29-8.34 (m), 7.80 (dd), 7.66 (dd), 7.22-7.33 (m), 7.13-7.21 (m),5.47-5.57 (m), 5.36-5.44 (m), 4.43-4.93 (m), 3.97-4.05 (m), 3.64-3.83(m), 3.32-3.61 (m), 3.15-3.29 (m), 3.06-3.09 (m), 2.95-3.05 (m),2.89-2.95 (m), 2.82-2.84 (m), 2.67-2.72 (m), 2.54-2.56 (m), 2.48-2.53(m), 2.28-2.48 (m), 2.11-2.28 (m), 2.09 (s), 1.57-1.72 (m), 1.38-1.47(m), 1.15-1.37 (m), 1.09 (dd), 0.85-0.99 (m), 0.78 (t).

Preparation of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,formate salt (#200) and1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,formate salt (#201)

Step 1 Synthesis of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#195)

According to the general procedure D, from #6 (7.75 g, 21.6 mmol, 1eq.), #152 (3.88 g, 21.6 mmol, 1 eq.), dichloromethane (100 mL), HATU(9.8 g, 25.9 mmol, 1.2 eq.), and diisopropylethylamine (11.1 g, 86.4mmol, 4 eq.) was synthesized the crude desired material, which waspurified by silica gel chromatography (Gradient: 20 to 55% ethyl acetatein petroleum ether) to afford #195 (11.1 g, quantitative yield) as ayellow oil.

Step 2 Synthesis of1,2-dimethyl-D-prolyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide(#196)

According to the general procedure B, from #195 (11.1 g, 21.6 mmol, 1eq.), dichloromethane (100 mL) and trifluoroacetic acid (40 mL) wassynthesized the crude desired material, to obtain #196 (10.1 g,quantitative yield) which was used in the next step without furtherpurification. LC-MS (Protocol Z): m/z 428.5 [M+H⁺], retention time=0.9minutes.

Step 3 Synthesis of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-5-methyl-1-oxo-1-(pentafluorophenoxy)heptan-4-yl]-N-methyl-L-valinamide(#197)

To a cooled solution (0° C.) of #196 (4.0 g, 9.4 mmol, 1 eq.) indichloromethane (40 mL) was added dropwise pyridine (2.95 g, 37.6 mmol,4 eq.) followed by a solution of pentafluorophenyl trifluoroacetate (3.9g, 13.6 mmol, 1.4 eq.) in dichloromethane (5 mL). The mixture wasstirred at room temperature for one hour, and the solvent wasconcentrated in vacuo. The residue was purified by silica gelchromatography (Gradient: 1 to 10% methanol in dichloromethane) toafford compound #197 (4.5 g, 81.2% (over three steps) as white solid.

Step 4 Synthesis of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#198)

To a cooled solution (0° C.) of #197 (4.0 g, 7.4 mmol, 1 eq.) indichloromethane (25 mL) was added dropwise diisopropylethylamine (3.4 g,26.3 mmol, 3.5 eq.) followed by a solution #103 (2.3 g, 7.6 mmol, 1.02eq.) in dichloromethane (15 mL). After the addition, the mixture wasstirred at room temperature for 16 hours and the solvent was removed invacuo. The residue was purified by silica gel chromatography (Gradient:1 to 10% methanol in dichloromethane) followed by a second purificationby reverse phase chromatography (Method Q) to give #198 (1.57 g, 57.5%)as white solid HPLC (Protocol X): m/z 597.49 [M+H⁺] retention time=8.879minutes (purity=98%). Chiral HPLC retention time: 3.328 min (purity=98%)Column: Column: Chiralcel OJ-H, 250×4.6 mm, 5 μm; Mobile phase: methanol(0.05% diethylamine) in CO₂ from 5% to 40% over 15 minutes; Flow rate:2.35 mL/minute.

Step 5 Synthesis of 1,2-dimethyl-D-prolyl-N-[(3R,45, 5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-(pentafluorophenoxy)propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#199)

To a solution of #198 (280 mg, 0.394 mmol, 1 eq.) in dichloromethane (2mL) was added pyridine (75 mg, 0.94 mmol, 2.4 eq.) followed by asolution of pentafluorophenyl trifluoroacetate (268 mg, 0.94 mmol, 2.4eq.) in dichloromethane (1.5 mL). The mixture was stirred at roomtemperature for 2.5 hours, and the solvent was concentrated in vacuo.The residue was purified by silica gel chromatography (Gradient: 1 to10% methanol in dichloromethane) to afford compound #199 (279 mg, 39%)as white solid. LC-MS (Protocol Q1): m/z 763.5 [M+H⁺], retentiontime=0.93 minutes.

Step 6A Synthesis of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-(1,2,3,4-tetrahydroquinolin-6-yl)propan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#200)

To a mixture of #199 (25 mg, 0.033 mmol, 1 eq) and #215 ((7.7 mg, 0.033mmol, 1 eq.) in dichloromethane (1.5 mL), N,N-diisopropylethylamine(30.2 mg, 2.31 mmol, 7 eq.) was added. The reaction was stirred for 5minutes and N,N-dimethylformamide (0.5 mL) was added. After stirring for2½ hours, additional N,N-diisopropylethylamine (30.2 mg, 2.31 mmol, 7eq.) was added. After 3½ hours, more N,N-dimethylformamide (0.75 mL) wasadded and the mixture stirred for 18 hours. Additional N,N-diisopropylethylamine (15.1 mg, 1.15 mmol, 3.6 eq.) andN,N-dimethylformamide (0.25 mL) were added and reaction stirred at roomtemperature for 48 hours. The reaction mixture was concentrated in vacuoand the crude product was purified by reverse phase chromatography(Method M*) to give #200 (12.9 mg, 48%). HPLC (Protocol T): m/z HPLC(Protocol 7): m/z 407.7, double charge [2+], retention time=1.69 minutes(purity=100%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.72-9.82 (m), 8.61-8.67 (m),8.42-8.48 (m), 8.19-8.24 (m), 7.25-7.27 (m), 7.12-7.14 (m), 6.94-7.01(m), 6.88-6.94 (m), 6.78-6.84 (m), 6.67-6.74 (m), 6.57-6.64 (m),4.69-4.77 (m), 4.60-4.68 (m), 4.53-4.60 (m), 4.46-4.53 (m), 4.37-4.45(m), 3.97-4.05 (m), 3.76-3.81 (m), 3.62-3.67 (m), 3.53-3.62 (m),3.44-3.52 (m), 3.32-3.38 (m), 3.27-3.32 (m), 3.22-3.27 (m), 3.15-3.22(m), 3.06-3.14 (m), 2.97-3.01 (m), 2.92-2.96 (m), 2.74-2.83 (m),2.61-2.74 (m), 2.57-2.61 (m), 2.48-2.56 (m), 2.37-2.46 (m), 2.25-2.36(m), 2.00-2.20 (m), 1.67-1.91 (m), 1.47-1.60 (m), 1.37-1.47 (m),1.24-1.35 (m), 1.03-1.10 (m), 0.95-1.00 (m), 0.88-0.94 (m), 0.76-0.83(m).

Step 6B Synthesis of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#201)

To a mixture of #199 (25 mg, 0.033 mmol, 1 eq) and the commerciallyavailable methy14-amino-L-phenylalaninate (8.8 mg, 0.033 mmol, 1 eq.) indichloromethane (1.5 mL), N,N-diisopropylethylamine (30.2 mg, 2.31 mmol,7 eq.) was added. The reaction was stirred for 5 minutes andN,N-dimethylformamide (0.5 mL) was added. After 4 hours, additionalN,N-diisopropylethylamine (37.75 mg, 2.88 mmol, 8.25 eq.) was added andthe mixture stirred for 50 minutes. Additional N,N-dimethylformamide(0.75 mL) was added and the reaction was stirred for 66 hours,concentrated in vacuo and the crude product was purified by reversephase chromatography (Method M*) to give #201 (14.3 mg, 56%); HPLC(Protocol T): m/z 387.2, double charge [2+], retention time=1.50 minutes(purity=100%); ¹H NMR (400 MHz, DMSO-d₆) δ 9.68-9.84 (m), 8.57-8.66 (m),8.46-8.51 (m), 8.23-8.29 (m), 7.18-7.28 (m), 7.11-7.16 (m), 7.03-7.08(m), 6.97-7.02 (m), 4.67-4.75 (m), 4.58-4.66 (m), 4.34-4.57 (m),3.95-4.03 (m), 3.85-3.90 (m), 3.73-3.81 (m), 3.66-3.72 (m), 3.57-3.66(m), 3.50-3.57 (m), 3.42-3.49 (m), 3.32-3.38 (m), 3.14-3.30 (m),2.95-3.11 (m), 2.84-2.94 (m), 2.78-2.81 (m), 2.63-2.74 (m), 2.46-2.57(m), 2.34-2.45 (m), 2.19-2.34 (m), 1.97-2.19 (m), 1.67-1.90 (m),1.45-1.62 (m), 1.34-1.41 (m), 1.21-1.34 (m), 1.01-1.10 (m), 0.82-0.99(m), 0.72-0.81 (m).

Preparation of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-(1,2,3,4-tetrahydroquinolin-6-yl)propan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,formate salt. (#207) and1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamideformate salt (#208) and1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#209)

Step 1 Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-tert-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#202)

According to the general procedure D, from #6 (4.3 g, 12 mmol, 1 eq.),#150 (2.15 g, 12 mmol, 1 eq.), dichloromethane (50 mL), HATU (5.46 g, 14mmol, 1.2 eq.), and diisopropylethylamine (8.17 mL) was synthesized thecrude desired material, which was purified by silica gel chromatography(Gradient: 20 to 55% ethyl acetate in petroleum ether) to afford #202(5.2 g, 89%) as a yellow oil. 1

Step 2 Synthesis of1,2-dimethyl-L-prolyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide(#203)

According to the general procedure B, from #202 (5.2 g, 10.77 mmol, 1eq.), dichloromethane (45 mL), and trifluoroacetic acid (20 mL) wassynthesized the crude desired material, to obtain #203 (7 g,quantitative yield) which was used in the next step without furtherpurification.

Step 3 Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-5-methyl-1-oxo-1-(pentafluorophenoxy)heptan-4-yl]-N-methyl-L-valinamide(#204)

To a cooled solution (0° C.) of #203 (7.0 g, 10.77 mmol, 1 eq.) indichloromethane (15 mL) was added dropwise pyridine (3.41 g 43.08 mmol,4 eq.) followed by a solution of pentafluorophenyl trifluoroacetate(6.03 g, 21.54 mmol, 2 eq.) in dichloromethane (7 mL). The mixture wasstirred at room temperature for one hour, and the solvent wasconcentrated in vacuo. The residue was purified by silica gelchromatography (Gradient: 1 to 10% methanol in dichloromethane) toafford compound #204 (8 g, 82% over two steps) as yellow solid.

Step 4 Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#205)

To a cooled solution (0° C.) of #204 (8.0 g, 10.77 mmol, 1 eq.) indichloromethane (25 mL) was added dropwise diisopropylethylamine (5.6 g,43.08 mmol, 4 eq.) followed by a solution of #103 (3.22 g, 10.77 mmol, 1eq.) in dichloromethane (15 mL). After the addition, the mixture wasstirred at room temperature for 16 hours and the solvent was removed invacuo. The residue was purified by silica gel chromatography (Gradient:1 to 10% methanol in dichloromethane) to give #205 (2.2 g, 33%) as ayellow solid HPLC (Protocol X): m/z 597.42 [M+H⁺], retention time=8.729minutes (purity >97%), Chiral HPLC retention time: 2.87 min (purity=89%)Column. Chiralcel OD-3, 150×4.6 mm, 3 nm; Mobile phase: ethanol (0.05%diethylamine) in CO₂ from 5% to 40% over 12 minutes; Flow rate: 2.5mL/minute.

Step 5 Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-(pentafluorophenoxy)propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#206)

To a solution of #198 (0.28 g, 0.47 mmol, 1 eq.) in dichloromethane (2mL) was added pyridine (75 mg, 0.94 mmol, 2 eq.) followed by a solutionof pentafluorophenyl trifluoroacetate (268 mg, 0.94 mmol, 2 eq.) indichloromethane (1.5 mL). The mixture was stirred at room temperaturefor 2.5 hours, and the solvent was concentrated in vacuo. The residuewas purified by silica gel chromatography (Gradient: 1 to 10% methanolin dichloromethane) to afford compound #206 (348 mg, 97%) as whitesolid. LC-MS (protocol Q1): m/z 763.5 [M+H⁺], retention time=0.9minutes.

Step 6A Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-(1,2,3,4-tetrahydroquinolin-6-yl)propan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt. (#207)

The title compound was prepared from #206 (25 mg, 0.033 mmol, 1 eq.) and#215 (7.7 mg, 0.033 mmol, 1 eq) using the method described above forpreparation of #200. The crude product was purified by reverse phasechromatography (Method M*) to give #207 (11.7 mg, 44%). HPLC (ProtocolT): m/z 407.6, double charge [2+], retention time=1.59 minutes(purity=100%). ¹H NMR (DMSO-d₆) δ 9.57-9.69 (m), 8.68-8.76 (m),8.42-8.47 (m), 8.23-8.29 (m), 8.18-8.23 (m), 7.24-7.27 (m), 6.95-7.01(m), 6.89-6.94 (m), 6.80-6.86 (m), 6.70-6.78 (m), 6.60-6.67 (m),4.69-4.77 (m), 4.60-4.68 (m), 4.46-4.60 (m), 4.34-4.46 (m), 3.95-4.03(m), 3.87-3.91 (m), 3.79-3.85 (m), 3.74-3.79 (m), 3.60-3.66 (m),3.49-3.60 (m), 3.41-3.49 (m), 3.12-3.35 (m), 3.04-3.12 (m), 2.89-3.04(m), 2.68-2.83 (m), 2.61-2.67 (m), 2.45-2.55 (m), 2.34-2.44 (m),2.08-2.33 (m), 2.05-2.08 (m), 1.92-2.05 (m), 1.75-1.91 (m), 1.65-1.75(m), 1.59-1.64 (m), 1.34-1.58 (m), 1.20-1.31 (m), 1.01-1.09 (m),0.94-0.99 (m), 0.84-0.93 (m), 0.80-0.83 (m), 0.72-0.80 (m).

Step 6B Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-aminophenyl)-1-methoxy-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamidetrifluoroacetic acid salt (#208)

To a mixture of #206 (25.0 mg, 0.033 mmol, 1 eq.), and the commerciallyavailable methy14-amino-L-phenylalaninate (8.8 mg, 0.033 mmol, 1 eq.) indichloromethane (1.5 mL), N,N-diisopropylethylamine (30.2 mg, 2.31 mmol,7 eq.) was added.

The reaction was stirred for 5 minutes and N,N-dimethylformamide (0.5mL) was added. After 2½ hours, additional N,N-diisopropylethylamine(30.2 mg, 2.31 mmol, 7 eq.) was added. After stirring for 3½ hours,additional N,N-dimethylformamide (0.75 mL) was added and The reactionwas stirred for 66 hours, concentrated in vacuo and the crude productwas purified by reverse phase chromatography (Method M*) to give #208(13 mg, 51%); HPLC (Protocol T): m/z 387.2, double charge [2+],retention time=1.58 minutes (purity=100%); ¹H NMR (400 MHz, DMSO-d₆) δ9.54-9.69 (m), 8.68-8.75 (m), 8.46-8.50 (m), 8.33-8.37 (m), 8.22-8.31(m), 8.09-8.14 (m), 7.17-7.27 (m), 7.07-7.16 (m), 6.99-7.05 (m),6.92-6.99 (m), 4.69-4.75 (m), 4.60-4.68 (m), 4.42-4.59 (m), 4.34-4.42(m), 3.95-4.03 (m), 3.85-3.90 (m), 3.74-3.80 (m), 3.65-3.72 (m),3.62-3.65 (m), 3.42-3.62 (m), 3.31-3.36 (m), 3.24-3.30 (m), 3.11-3.24(m), 3.03-3.11 (m), 2.96-3.03 (m), 2.81-2.92 (m), 2.65-2.76 (m),2.43-2.55 (m), 2.34-2.43 (m), 2.06-2.33 (m), 1.93-2.05 (m), 1.75-1.89(m), 1.66-1.74 (m), 1.58-1.65 (m), 1.47-1.57 (m), 1.34-1.43 (m),1.20-1.32 (m), 1.10-1.14 (m), 1.00-1.09 (m), 0.83-0.99 (m), 0.72-0.81(m).

Step 6C Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#209)

The title compound was prepared from #206 (25.0 mg, 0.033 mmol, 1 eq.)and methyl -L-phenylalaninate hydrochloride (7.1 mg, 0.033 mmol, 1 eq.)using the method described above for preparation of #200. The crudeproduct was purified by reverse phase chromatography (Method M*) to give#209 (10.3 mg, 41%). HPLC (Protocol T): m/z 758.7 [M+H⁺], retentiontime=1.787 minutes (purity=100%). ¹H NMR (DMSO-d₆) δ 9.57-9.70 (m),8.70-8.75 (m), 8.50-8.56 (m), 8.32-8.42 (m), 8.24-8.26 (m), 8.10-8.15(m), 7.14-7.27 (m), 7.12-7.13 (m), 6.98-7.00 (m), 4.69-4.77 (m),4.55-4.67 (m), 4.43-4.53 (m), 3.94-4.02 (m), 3.73-3.78 (m), 3.62-3.68(m), 3.48-3.60 (m), 3.39-3.48 (m), 3.23-3.33 (m), 3.13-3.22 (m),3.08-3.13 (m), 3.02-3.08 (m), 2.96-3.01 (m), 2.81-2.94 (m), 2.76-2.80(m), 2.66-2.75 (m), 2.62-2.66 (m), 2.46-2.55 (m), 2.31-2.45 (m),2.09-2.29 (m), 2.05-2.09 (m), 1.93-2.04 (m), 1.74-1.88 (m), 1.65-1.74(m), 1.59-1.65 (m), 1.36-1.52 (m), 1.21-1.35 (m), 1.01-1.08 (m),0.94-1.00 (m), 0.83-0.94 (m), 0.73-0.81 (m).

Preparation of methyl(2S)-2-amino-3-(1,2,3,4-tetrahydroquinolin-6-yl)propanoate

Step 1 Synthesis of 6-(bromomethyl)quinoline (#210)

A solution of 6-methylquinoline (5 g, 35 mmol, 1 eq.),N-Bromosuccinimide (8.1 g, 45.5 mmol, 1.3 eq.) and benzoyl peroxide (840mg, 3.5 mmol, 0.1 eq.) in carbon tetrachloride (100 mL) was stirred atreflux for 3 hours and then cooled to room temperature. The reactionmixture was filtered and the filtrate was concentrated in vacuo. Theresidue was dissolved in tetrahydrofuran (100 mL) and filtered. Thefiltrate was directly used in the next step without further purification

Step 2 Synthesis of6-{[(2S,5R)-3,6-dimethoxy-5-(propan-2-yl)-2,5-dihydropyrazin-2-yl]methyl}quinoline(#211)

To a cooled solution (−70° C.) of(2R)-3,6-dimethoxy-2-(propan-2-yl)-2,5-dihydropyrazine (25.8 g, 140mmol, 2 eq.) in tetrahydrofuran (200 mL) was added dropwisen-butyllithium (2.5 M, 64.4 mL, 161 mmol 2.3 eq.) and then stirred for30 minutes. A solution of #210 (15.4 g, 70 mmol, 1 eq.) intetrahydrofuran (150 mL) was added dropwise at −65° C. and then thesolution was stirred for 2 hours at this temperature. The reaction wasquenched by saturated aqueous ammonium chloride (100 mL) and extractedwith ethyl acetate (100 mL). The organic phase was dried over sodiumsulfate and concentrated in vacuo. The residue was purified by silicacolumn chromatography (Gradient: 10 to 16% ethyl acetate in petroleumether) to afford #211 (7.3 g, 32%, over two steps) as yellow solid.LC-MS (Protocol Z): m/z 326.2 [M+H⁺], retention time=0.88 minutes.

Step 3 Synthesis of methyl (2S)-2-amino-3-(quinolin-6-yl)propanoate(#212)

To a solution of #211 (7.3 g, 22.5 mmol, 1 eq.) in water (25 mL) andacetonitrile (80 mL) was added trifluoroacetic acid (9 mL) at 0° C. andthe solution was stirred at 10° C. overnight. The organic layer wasremoved in vacuo and the remaining aqueous layer was basified to pH 9with sodium carbonate, which was directly used for the next step.

Step 4 Synthesis of methyl(2S)-2-[(tert-butoxycarbonyl)amino]-3-(quinolin-6-yl)propanoate (#213)

To a solution #212 (5.2 g, 22.5 mmol, 1 eq.) and triethylamine (9.1 g,90 mmol, 4 eq.) in mixed solvent of methanol (30 mL) and water (50 mL)was added di-tert-butyl dicarbonate (17.5 g, 78.75 mmol, 3.5 eq.) at 0°C. and then the solution was stirred at 10° C. overnight. The reactionmixture was filtered and the filter cake was washed with methanol (20mL×2). The filtrate was extracted with ethyl acetate (50 mL×2) and theorganic phase was concentrated in vacuo. The residue was purified bysilica column chromatography (Gradient: 25 to 50% ethyl acetate inpetroleum ether) to afford #213 (5.5 g, 74% over two steps) as yellowoil. LC-MS (Protocol Z): m/z 331.2 [M+H⁺], retention time=0.76 minutes.

Step 5 Synthesis of methyl(2S)-2-[(tert-butoxycarbonyl)amino]-3-(1,2,3,4-tetrahydroquinolin-6-yl)propanoate(#214)

A suspension of #213 (1.5 g, 4.55 mmol, 1 eq.) and palladium on carbon(150 mg) in ethanol (20 mL) was stirred at 50° C. under 30 psi ofhydrogen overnight. The reaction mixture was filtered through a pad ofcelite and the filtrate was concentrated in vacuo. The residue waspurified by silica column chromatography (Gradient: 40% ethyl acetate inpetroleum ether) to afford #214 (1.2 g, 80%) as an oil. ¹H NMR (400 MHz,CDCl₃): δ 6.70 (d, 2H), 6.40 (m, 1H), 4.95 (m, 1H), 4.49 (m, 1H), 3.77(s, 3H), 3.28 (m, 2H), 2.97 (m, 2H), 2.71 (m, 2H), 1.95 (m, 2H), 1.26(s, 9H), HPLC (Protocol Y): m/z 357.0 [M+Na⁺] retention time=5.304minutes (purity >98%). Chiral HPLC retention time: 4.64 min(purity=98%)(Column: Chiralcel OJ-H, 150×4.6 mm, 5 μm; Mobile phase:ethanol (0.05% diethylamine) in CO₂ from 5% to 40% over 15 minutes; Flowrate: 2.5 mL/minute.

Step 6 Synthesis of methyl(2S)-2-amino-3-(1,2,3,4-tetrahydroquinolin-6-yl)propanoate (#215)

To a solution of #214 (750 mg, 2.25 mmol, 1 eq.) in dichloromethane (20mL) was added dropwise trifluoracetic acid (2 mL) at 0° C. and then thesolution was stirred at 20° C. overnight. The reaction mixture wasconcentrated in vacuo and the residue was dissolved in water (20 mL).The solution was basified with sodium carbonate and extracted with ethylacetate/tetrahydrofuran (30 mL×3). The organic phase was dried oversodium sulfate and concentrated in vacuo to afford #215 (450 mg, 85%) asa yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 6.70 (d, 2H), 6.40 (m, 1H),3.73 (s, 3H), 3.67 (m, 1H), 3.30 (m, 2H), 2.96 (m, 1H), 2.75 (m, 3H),1.96 (m, 2H), 1.50 (br, 2H), 1.26 (br, 1H), HPLC (Protocol Y): m/z235.14 [M+H⁺] retention time=4.35 minutes (purity >96%). Chiral HPLCretention time: 5.71 min (purity=98%). (Column: Chiralcel OJ-H, 150×4.6mm, 5 μm; Mobile phase: ethanol (0.05% diethylamine) in CO₂ from 5% to40% over 15 minutes; Flow rate: 2.5 mL/minute.

Preparation ofN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)anaino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt (#217) andN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt (#219)

Step 1A Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate(#216)

To a solution of #168 (36.9 mg, 0.158 mmol, 1 eq.) and #114 (100 mg,0.158 mmol, 1 eq.) in dichloromethane (3.6 mL) and N,N-dimethylformamide(0.8 mL), was added diisopropylethylamine (0.083 mL, 0.474 mmol, 3 eq.)followed by HATU (60.7 mg, 0.158 mmol, 1 eq.). The reaction was allowedto stir at room temperature for 18 hours, diluted with ethyl acetate (25mL), washed with water (1×), 10% citric acid (1×) and brine (1×). Theorganic layer was dried over sodium sulfate, filtered, and the filtrateconcentrated in vacuo to give crude #216 (220 mg, 164% of theory) whichwas used in next step without further purification. HPLC (protocol Q):m/z 848.6 [M+H⁺], retention time=2.10 minutes.

Step 1B Synthesis of methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3S)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate(#218)

To a solution of #169 (36.9 mg, 0.158 mmol, 1 eq.) and #114 (100 mg,0.158 mmol, 1 eq.) in dichloromethane (3.6 mL) and N,N-dimethylformamide(0.8 mL), was added diisopropylethylamine (0.083 mL, 0.474 mmol, 3 eq.)followed by HATU (60.7 mg, 0.158 mmol, 1 eq.). The reaction was allowedto stir at room temperature for 18 hours, diluted with ethyl acetate (25mL), washed with water (1×), 10% citric acid (1×) and brine (1×). Theorganic layer was dried over sodium sulfate, filtered, and the filtrateconcentrated in vacuo to provide crude #218 (180 mg, 134% of theory)which was used in next step without further purification. HPLC (protocolQ): m/z 848.6 [M+H⁺], retention time=2.10 minutes.

Step 2A Synthesis ofN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-{[(3R)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt (#217)

To a solution of crude #216 (134 mg) in tetrahydrofuran (4 mL) was addedlithium hydroxide (1M, 0.5 mL). The reaction was stirred at roomtemperature for 18 hours and concentrated in vacuo. The residue wasdissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) wasadded. The reaction was stirred for 4 hours and concentrated in vacuo.The crude material was purified by reverse phase chromatography (MethodM*) to obtain #217 (60 mg, 86% over two steps) as a gum. LC-MS (protocolQ): m/z 734.93 [M+H⁺], retention time=1.19 minutes. ¹H NMR (DMSO-d₆)12.62-12.83 (m), 9.30-9.43 (m), 9.17-9.28 (m), 8.34-8.41 (m), 8.22-8.31(m), 8.08-8.15 (m), 7.87-7.93 (m), 7.76-7.81 (m), 7.11-7.23 (m),4.93-4.99 (m), 4.81-4.88 (m), 4.55-4.71 (m), 4.48-4.54 (m), 4.37-4.45(m), 3.92-3.99 (m), 3.69-3.75 (m), 3.31-3.65 (m), 3.25-3.30 (m),3.20-3.24 (m), 3.12-3.19 (m), 3.08-3.10 (m), 2.97-3.07 (m), 2.92-2.97(m), 2.75-2.84 (m), 2.64-2.70 (m), 2.43-2.57 (m), 2.28-2.43 (m),2.15-2.26 (m), 2.02-2.15 (m), 1.56-1.87 (m), 1.31-1.48 (m), 1.05-1.30(m), 0.97-1.06 (m), 0.82-0.97 (m), 0.71-0.79 (m).

Step 2B Synthesis ofN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N-[(3S)-3-fluoropyrrolidin-3-yl]carbonyl}-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanine,trifluoroacetic acid salt (#219)

To a solution of crude #218 (100 mg) in tetrahydrofuran (4 mL) was added1.0 M lithium hydroxide in water (0.5 mL). The reaction was stirred atroom temperature for 18 hours and then concentrated in vacuo. The crudematerial was dissolved in dichloromethane (2 mL) and trifluoroaceticacid (2 mL) was added. The reaction was stirred for 4 hours and thenconcentrated in vacuo. The crude material was purified by reverse phasechromatography (Method M*) to obtain #219 as a gum (60 mg, 91% over twosteps). LC-MS (protocol Q): m/z 734.97 [M+H⁺], retention time=1.14minutes. ¹H NMR (400 MHz, DMSO-d₆), δ 12.62-12.85 (m), 9.32-9.43 (m),9.13-9.26 (m), 8.39-8.46 (m), 8.30-8.39 (m), 8.25-8.29 (m), 8.08-8.13(m), 7.79-7.85 (m), 7.67-7.72 (m), 7.10-7.23 (m), 4.94-5.01 (m),4.83-4.89 (m), 4.64-4.73 (m), 4.56-4.63 (m), 4.44-4.50 (m), 4.37-4.44(m), 3.92-3.99 (m), 3.60-3.74 (m), 3.24-3.55 (m), 3.11-3.24 (m),3.07-3.10 (m), 3.02-3.06 (m), 2.98-3.02 (m), 2.93-2.97 (m), 2.75-2.85(m), 2.68-2.69 (m), 2.63-2.67 (m), 2.45-2.55 (m), 2.26-2.44 (m),2.15-2.25 (m), 2.03-2.14 (m), 1.55-1.87 (m), 1.31-1.47 (m), 1.15-1.31(m), 0.98-1.05 (m), 0.91-0.98 (m), 0.82-0.91 (m), 0.71-0.78 (m).

Preparation of2-methylalanyl-N-{(3R,4S,5S)-1-[(2S)-2-{(3R,4R,7S)-7-benzyl-4-methyl-18-[(4S,5R)-5-methyl-2-oxoimidazolidin-4-yl}-5,8,13-trioxo-2-oxa-6,9,12-triazaoctadecan-3-yl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#257)

Step 1 Synthesis of 9H-fluoren-9-ylmethyl [(2S)-1-({2-[(tertbutoxycarbonyl)amino]ethyl}amino)-1-oxo-3-phenylpropan-2-yl]carbamate(#253)

Following general procedure D usingN-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-phenylalanine (500 mg, 1.29 mmol,1.0 eq), tert-butyl (2-aminoethyl)carbamate (207 mg, 1.29 mmol, 1.0eq.), HATU (620 mg, 1.55 mmol, 1.2 eq.) and Hunig's base (0.452 mL, 2.58mmol, 2.0 eq) in 6 mL of DMF #253 was yielded as a white solid (620 mg,91%) following concentration of solvent and re-crystallization usingethyl acetate. LC-MS (Protocol Q1): m/z 552.3 [M+Na⁺], retentiontime=1.01 minutes.

Step 2 Synthesis ofN-alpha-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-[2-({6-[(4S,5R)-5-methyl-2-oxoimidazolidin-4-yl]hexanoyl}amino)ethyl]-L-phenylalaninamide(#254)

Boc protection was removed using general procedure C using #251 (88 mg,0.17 mmol, 1.0 eq.) and 4M HCl (2.0 mL, 8.0 mmol, 48 eq.) followed byconcentration in vacuo. Coupling reaction was then performed followinggeneral procedure D using crude residue,6-[(4S,5R)-5-methyl-2-oxoimidazolidin-4-yl]hexanoic acid (35.6 mg, 0.166mmol, 1.0 eq.), HATU (73.2 mg, 0.18 mmol, 1.1 eq.), and Hunig's base(0.087 mL, 0.50 mmol, 3.0 eq.) in 2 mL of DMF followed by purification(Method J) yielding #254 (35 mg, 34%) as a white solid. LC-MS (ProtocolQ1): m/z 626.3 [M+H⁺], retention time=0.86 minutes.

Step 3 Synthesis ofN-[2-{6-[(4S,5R)-5-methyl-2-oxoimidazolidin-4-yl]hexanoyl}amino)ethyl]-L-phenylalaninamide(#255)

Following general procedure A using #254 (35 mg, 0.056 mmol, 1.0 eq.),piperidine (0.10 mL, 1.0 mmol, 20 eq.) in 0.5 mL of DMF followed bypurification using silica chromatography (0-30% methanol indichloromethane) affords #253 (19 mg, 84%). LC-MS (Protocol Q1): m/z404.2 [M+H⁺], retention time=0.48 minutes.

Step 4 Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-{(3R,4S,5S)-1-[(2S)-2-{(3R,4R,7S)-7-benzyl-4-methyl-18-[(4S,5R)-5-methyl-2-oxoimidazolidin-4-yl}-5,8,13-trioxo-2-oxa-6,9,12-triazaoctadecan-3-yl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#256)

Following general procedure D using #105 (36.6 mg, 0.047 mmol, 1.0 eq.),#255 (19 mg, 0.047 mmol, 1.0 eq.), HATU (22.4 mg, 0.056 mmol, 1.2 eq.)and Hunig's base (0.025 mL, 0.141 mmol) in 1.5 mL of DMF following bypurification (Method J) yielded #256 (15 mg, 27%) as a white solid.LC-MS (Protocol Q1): m/z 1164.8 [M+H⁺], retention time=0.99 minutes.

Step 5 Synthesis of2-methylalanyl-N-{(3R,4S,5S)-1-[(2S)-2-{(3R,4R,7S)-7-benzyl-4-methyl-18-[(4S,5R)-5-methyl-2-oxoimidazolidin-4-yl]-5,8,13-trioxo-2-oxa-6,9,12-triazaoctadecan-3-yl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide(#257)

Following general procedure A using #256 (5 mg, 0.004 mmol, 1.0 eq.) andpiperidine (0.02 mL, 0.2 mmol, 50 eq.) in 0.7 mL of DMF followed bypurification (Method J) afforded #257 (2 mg, 50%) as a colorless glass.LC-MS (Protocol Q1): m/z 1164.8 [M+H⁺], retention time=0.99 minutes. ¹HNMR (400 MHz, DMSO-d₆), δ 8.44-8.52 (m), 8.06-8.20 (m), 7.96-8.01 (m),7.69-7.83 (m), 7.20-7.28 (m), 7.11-7.19 (m), 3.38-3.83 (m), 3.19-3.26(m), 3.03-3.12 (m), 2.98 (s), 2.91 (s), 2.75 (s), 2.65-2.70 (m),2.01-2.36 (m), 1.65-1.87 (m), 1.39-1.57 (m), 1.13-1.37 (m), 1.04-1.08(m), 0.74-1.01 (m).

Preparation ofN-[5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(mv#115)

Step 1 Preparation ofN-[5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(mv#115)

To a stirring solution of5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoic acid (12 mg, 0.061 mM)in 0.4 mL of dichloromethane, and 0.1 mL of DMF, HATU (23.2 mg, 0.061mM) was added followed by Hunig's base (0.033 mL, 0.188 mM). Thereaction was allowed to stir for 5 minutes before #115 (39 mg, 0.047 mM)was added as a solution in 0.4 mL of dichloromethane, and 0.1 mL of DMF.The reaction was allowed to stir at room temperature for 3 hours and 15minutes before being quenched through the addition of water containing asmall amount of TFA. Reaction was then reduced down. Crude material wasdissolved with DMSO and purified by reverse phase chromatography (MethodJ). The appropriate fractions were concentrated then (Genevac). Materialwas then further purified by reverse phase chromatography (Method K)with the appropriate fractions being concentrated (Genevac). Materialwas then transferred to a small vial using dichloromethane and methanolbefore being reduced down (Genevac) to afford mv#115 (1.4 mg, 3.3%)oil/solid mix. HPLC (Protocol A at 45° C.): m/z 897.5 [M+H⁺], retentiontime=9.149 minutes (purity >97%).

Preparation ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(mc#115)

Step 1 Synthesis of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylchloride (#248)

To a stirring solution of6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (3.15 g, 14.9 mM)in 15 mL of dichloromethane, oxalyl chloride (1.61 mL, 17.9 mM) wasadded followed by one drop of DMF. The reaction was allowed to stir atroom temperature for three hours. The reaction was concentrated invacuo. The residue was dissolved in a one to one solution of heptane anddichloromethane and then concentrated in vacuo. This process wasrepeated two more times producing a solid #248 (3.43 g, 100%). ¹H NMR(400 MHz, DMSO-d₆): δ [7.02 (s, 2H), 3.43 (m, 2H), 2.53 (m, 1H),CH_(2.18) (m, 1H), 1.54 (m, 4H), 1.26 (m, 2H).]

Step 2 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N,2-dimethylalanine(#249)

To a stirring solution of #248 (600 mg, 2.61 mM) in 10 mL ofdichloromethane, N,2-dimethylalanine (306 mg, 2.61 mM) was addedfollowed by triethylamine (1.09 mL, 7.84 mM). The reaction was allowedto stir at room temperature for three hours. Dichloromethane was addedto the reaction and the organic layer was washed three times with waterand two times with brine. The organic layer was separated and then driedover sodium sulfate before being concentrated in vacuo. The cruderesidue was purified by silica chromatography (0-30% methanol indichloromethane) on silica which had been previously neutralized withtriethylamine yielding a white solid #249 (127 mg, 16%). LC-MS (ProtocolQ): m/z 309.0 [M−H⁻], retention time=0.96 minutes.

Step 3 Synthesis ofN-{(2R,3R)-3-methoxy-3-[(2S)-1-{(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoyl}pyrrolidin-2-yl]-2-methylpropanoyl}-L-phenylalanine(#250)

To a stirring solution of #113 (2.10 g, 2.46 mM) in 10 mL of THF,lithium hydroxide (228 mg, 5.16 mM) was added followed by 3 mL of water.The reaction was allowed to stir at room temperature for 2 hours. Thereaction was acidified though the addition of 1 M HCl and thenconcentrated in vacuo. The resulting white solid was taken up in 20 mLof acetonitrile and mL of water. The aqueous layer removed and theorganic layer was washed once with water. The organic layer was driedover sodium sulfate, filtered, and concentrated in vacuo. Ethyl acetate(20 mL) was then added and the crude solid was allowed to stir for 30minutes, before being filtered to yield a white solid #250 (1.42 g,94%). LC-MS (Protocol Q): m/z 619.5 [M+H⁺], retention time=1.10 minutes.HPLC (Protocol A at 45° C.) m/z 619.4 [M+H⁺], retention time=6.732minutes.

Step 4 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(mc#115)

To a stirring solution of #249 (382 mg, 1.23 mM) in 5 mL ofdichloromethane, HATU (482 mg, 1.23 mM) was added followed bytriethylamine (0.52 mL, 1.23 mM). The reaction was allowed to stir for 1hour at room temperature followed by the addition of #250 (762 mg, 1.23mM). The reaction was allowed to stir for 3 hours. Reaction wasconcentrated in vacuo. Reverse phase purification (MethodL) followed bylyophilization yielded a white solid mc#120 (124 mg, 11%). HPLC(Protocol A at 45° C.;) m/z 911.5 [M+H⁺], retention time=9.676 minutes.

Preparation ofN-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(mb#115)

Step 1 Synthesis ofN-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(mb#115)

A stirring solution of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoicacid (1.2 equivalents), HATU (1.2 equivalents), and Hunig's base (3equivalents) in DMF and dichloromethane is allowed to stir for 30minutes. Compound #115 (1 equivalent) is then added as a solution indichloromethane and DMF. Reaction is monitored by LC-MS. Reaction isconcentrated down and purification is completed by Isco medium pressurereverse phase chromatography (Gradient: 5%-100% water in acetonitrile).

Preparation ofN-[7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)heptanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(me#115)

Step 1 Synthesis ofN-[7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)heptanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(me#115)

A stirring solution 7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)heptanoicacid (1.2 equivalents), HATU (1.2 equivalents), and Hunig's base (3equivalents) in DMF and dichloromethane is allowed to stir for 30minutes. Compound #115 (1 equivalent) is then added as a solution indichloromethane and DMF. Reaction is monitored by LC-MS. Reaction isconcentrated down and purification is completed by Isco medium pressurereverse phase chromatography (Gradient: 5%-100% water in acetonitrile).

Preparation ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yOhexanoylPL-valyl-N˜5˜-carbamoyl-N-(4-{(8S,11S,12R)-[2-(2-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-8-isopropyl-4,5,5,10-tetramethyl-11-[(1S)-1-methylpropyl]-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradec-1-yl}phenyl)-L-ornithinamide(mcValCitPABC#115)

Step 1 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N˜5˜-carbamoyl-N-(4-{(8S,11S,12R)-12-(2-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-8-isopropyl-4,5,5,10-tetramethyl-11-[(1S)-1-methylpropyl]-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradec-1-yl}phenyl)-L-ornithinamide(mcValCitPABC#115)

A solution of mcCValCitPABC (Linker # D, 1 equivalent) and #115 (1equivalent) in DMF is prepared. Hunig's base (4 equivalents),2,6-Luditine (4 equivalents), and HOAT (0.2 equivalents) is added.Reaction is monitored by LC-MS. Reaction is concentrated down andpurification is completed by Isco medium pressure reverse phasechromatography (Gradient: 5%-100% water in acetonitrile).

Preparation ofN-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(AmPeg6C2#115)

Step 1 Synthesis ofN-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide(AmPeg6C2#115)

A solution of1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacosan-25-oicacid (1 equivalent), HATU (1 equivalent), and Hunig's base (3equivalents) is allowed to stir for 30 minutes. Compound #115 is addedas a solution in DMF. Reaction is monitored by LC-MS. When couplingreaction is near completion, piperidine (5 equivalents) is added. Fmocde-protection is monitored by LC-MS. Reaction is concentrated down andpurification is completed by Isco medium pressure reverse phasechromatography (Gradient: 5%-100% water in acetonitrile).

Preparation of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-3-[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycyl}amino)phenyl]-1-methoxy-1-oxopropan-2-yl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamidemcGly#201

Step 1 Synthesis of methylN-(tert-butoxycarbonyl)-4-({N-[6-(2,5-dioxo-2,5-dihydro-1Hpyrrol-1-yl)hexanoyl]glycyl}amino)-L-phenylalaninate (#251)

To a solution of methyl4-amino-N-(tert-butoxycarbonyl)-L-phenylalaninate (4.1 g, 15.3 mmol, 1eq.) in dry N,N-dimethylformamide (70 mL) was addedN,N′-Dicyclohexylcarbodiimide (2.9 g, 15.3 mmol, 1 eq.) at 0° C. Themixture was stirred at 0° C. for 30 minutes. A solution of2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)acetic acid (3 g,10.2 mmol, 0.66 eq.) in dry N,N-dimethylformamide (20 mL) was added at0° C. The mixture was stirred at room temperature for 3 days. Themixture was filtered. The filtrate was poured into ice water (200 mL)and extracted with EtOAc (200 mL×3). The extract was washed with brine(200 mL), dried over Na₂SO₄ and concentrated in vacuo to afford #251(1.8 g, 32.3% yield) as a light yellow solid. HPLC (Protocol Q2) [M+Na⁺]567.3, retention time=1.02 min

Step 2 Synthesis of methyl4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycyl}amino)-L-phenylalaninate(#252)

To a solution of #251 (800 mg, 1.47 mmol, 1 eq.) in dichloromethane (16mL) was added TFA (4.8 mL) at 0° C. The mixture was stirred at roomtemperature for 2 hours. The mixture was concentrated in vacuo. Theresidue was dissolved in water and filtered. The filtrate waslyophilized to afford #252 (800 mg, 97.5%) as a white solid. HPLC(Protocol Q3) [M+H⁺] 1445.4, retention time=0.90 min.

Step 3 Synthesis of1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-3-[4-({N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycyl}amino)phenyl]-1-methoxy-1-oxopropan-2-yl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(mcGly#201)

To a solution of #198 (94 mg, 0.13 mmol, 1 eq.) and #252 (60.3 mg, 0.18mmol, 1.4 eq.) in N,N-dimethylformamide (2 mL) was added HATU (64.2 mg,0.13 mmol, 1 eq.) followed by N,N-diisopropylethylamine (66 mg, 0.52mmol). The solution was stirred at room temperature for 1 hour. Thereaction mixture was neutralized with aq. critic acid and concentratedto give crude product, which was purified by silica gel chromotography(eluted with MeOH/DCM from 1% to 7%), then purified again by preparativeTLC (Methanol:dichloromethane: =1:10) to give mcGly#201 (25 mg, 16.2%)as a white solid ESI-MS: m/z 1023.59 [M+H⁺], HPLC (ProtocolEB) retentiontime=4.0 minutes (Purity=96%). ¹H NMR (DMSO-d₆) 9.88 (d, 1H), 8.48 (d,0.5H), 8.24 (d, 0.5H), 8.11 (m, 1H), 7.82 (m, 1H), 7.47 (d, 2H), 7.15(m, 2H), 7.01 (s, 2H), 4.67 (m, 3H), 3.96 (m, 4H), 3.65 (m, 4H), 3.40(m, 4H), 3.27 (m, 7H), 3.16 (m, 5H), 2.24 (m, 8H), 1.50 (m, 11H), 1.19(m, 21H).

Preparation of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]amino}-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(MalC6Am#151)

Step 1 Synthesis of1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]amino}-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(MalC6Am#151)

Following general procedure D using #151 (20 mg, 0.023 mmol, 1.0 eq.),1-(6-aminohexyl)-1H-pyrrole-2,5-dione (7.0 mg, 0.030 mmol, 1.3 eq.),HATU (11.4 mg, 0.030 mmol, 1.3 eq.), and Hunig's base (0.016 mL, 0.092mmol, 1.3 eq.) in 2 mL of dichloromethane, and 0.2 mL of DMF followed bypurification using medium pressure reverse phase C18 chromatography(Gradient: 5% to 80% acetonitrile in water with 0.02% TFA in each phase)yielded MalC6 μm#151 (18.4 mg, 86%) as a clear oil/solid mix. LC-MS(Protocol Q): m/z 922.3 [M+H⁺], retention time=1.43 minutes; HPLC(Protocol A at 45° C.): m/z 922.4 [M+H⁺], retention time=7.203 minutes.

Preparation ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{(6S,9R,10R)-6-benzyl-10-[(2S)-1-{(3R,4S,5S)-4-[(1,2-dimethyl-L-prolyl-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-9-methyl-3,8-dioxo-2,11-dioxa-4,7-diazadodec-1-yl}phenyl)-N˜5˜-carbamoyl-L-ornithinamide(mcValCitPABC#246)

Step 1 Synthesis ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-(4-{(6S,9R,10R)-6-benzyl-10-[(2S)-1-{(3R,4S,5S)-4-[(1,2-dimethyl-L-prolyl-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-9-methyl-3,8-dioxo-2,11-dioxa-4,7-diazadodec-1-yl}phenyl)-N˜5˜-carbamoyl-L-ornithinamide(mcValCitPABC#246)

Following general procedure E using #246 (29.2 mg, 0.035 mmol, 1.0 eq.),mcValCitPABC-PNP (28.8 mg, 0.039 mmol, 1.1 eq.), 2,6-Luditine (0.016 mL,0.14 mmol, 4.0 eq.), Hunig's base (0.025 mL, 0.14 mmol, 4.0 rq.), andHOAT (4.8 mg, 0.035 mmol, 1.0 eq.) in 2.0 mL of DMA followed bypurification using medium pressure reverse phase C18 chromatography(Gradient: 5% to 50% acetonitrile in water with 0.02% TFA in each phase)yielded mcValCitPABC#246 (21 mg, 45%) as a clear oil/solid mix. LC-MS(Protocol Q): m/z 1327.9 [M+H⁺], retention time=1.36 minutes.

HERCEPTIN® In Vitro and In Vivo Studies

It is noted that for the following studies HERCEPTIN® in the absence ofconjugated cytotoxic agents shows no significant in vitro potency or invivo efficacy at equivalent antibody concentrations.

In vitro Cell Assay Procedure

Target expressing (BT474 (breast cancer), N87 (gastric cancer), HCC1954(breast cancer), MDA-MB-361-DYT2 (breast cancer)) or non-expressing(MDA-MB-468) cells were seeded in 96-well cell culture plates for 24hours before treatment. Cells were treated with 3-fold serially dilutedantibody-drug conjugates or free compounds (i.e., no antibody conjugatedto the drug) in duplicate at 10 concentrations. Cell viability wasdetermined by CellTiter 96® AQ_(ueous) One Solution Cell ProliferationMTS Assay (Promega, Madison Wis.) 96 hours after treatment. Relativecell viability was determined as percentage of untreated control. IC₅₀values were calculated using a four parameter logistic model #203 withXLfit v4.2 (IDBS, Guildford, Surry, UK). Results are shown in Tables 20,21A and 21B.

In vivo MDAMB-361 DYT2 Tumor Xenograft Model

In vivo efficacy studies of antibody-drug conjugates were performed withthe Her2+MDAMB-361 DYT2 cell line. For efficacy studies, 10 milliontumor cells in 50% matrigel were implanted subcutaneously into 6-8 weekold irradiated nude mice. When the tumor sizes reached between 250-350mm³ drugs or vehicle were administered through bolus tail veininjection. Mice were injected with 1 mg/kg of antibody drug conjugatestreated four times every four days (Q4dx4). Tumor volume is measuredtwice a week for the first 50 days and once weekly thereafter by aCaliper device and calculated with the following formula: Tumorvolume=(length×width)/2. Results were compared across studies bynormalizing the tumor regression of the drug-treated mice by dividingthe tumor volume by the vehicle-treated tumor volume (T/C).

Six compounds were tested in the three different MDA-MB-361-DYT2xenograft studies to determine their anti-tumor activity. The results ofa representative study with four of the compounds demonstratessignificant tumor regression from the vehicle-treated mice over the 50day observation period (FIG. 1). To compare the results of compounds inthe three studies, anti-tumor activity was normalized by dividing thedrug-treated tumor volume by the vehicle-treated tumor volume (T/C). Aplot of the six T/C values (FIG. 2) demonstrates that each of the sixcompounds causes complete (or almost complete) tumor regression over theobservation period which was up to 107 days for one of the studies.

Results of the testing of H(C)-#D54, H(C)-vcMMAE, H(C)-mcMMAF andH(K)-MCC-DM1 in the MDA-MB-361-DYT2 xenograft studies are shown in FIG.4. Tumor volume in treatment group over control group (T/C) plot allowscomparison between conjugates (see FIG. 5C). These results demonstratethat H(C)-#D54 displays equivalent efficacy to HERCEPTIN® conjugateswith H(C)-vcMMAE, H(C)-mcMMAF and is superior to H(K)-MCC-DM1 in thismodel.

In Vivo N87 Tumor Xenograft Model (HERCEPTIN®)

In vivo efficacy studies of antibody-drug conjugates were performed withtarget-expressing xenograft models using the N87 cell lines. Forefficacy study, 7.5 million tumor cells in 50% matrigel are implantedsubcutaneously into 6-8 weeks old nude mice until the tumor sizes reachbetween 250 and 350 mm³. Dosing is done through bolus tail veininjection. Depending on the tumor response to treatment, animals areinjected with 1-10 mg/kg of antibody drug conjugates treated four timesevery four days. All experimental animals are monitored for body weightchanges weekly. Tumor volume is measured twice a week for the first 50days and once weekly thereafter by a Caliper device and calculated withthe following formula: Tumor volume=(length×width)/2 Animals arehumanely sacrificed before their tumor volumes reach 2500 mm³. The tumorsize is observed to decrease after the first week of treatment Animalsmay be monitored continuously for tumor re-growth after the treatmenthas discontinued.

Results of the testing of H(C)-#D54, H(C)-vcMMAE, H(C)-mcMMAF andH(K)-MCC-DM1 in the N87 mouse xenograft in vivo screening model areshown in FIGS. 3 and 5. These results demonstrate that H(C)-#D54 issuperior/similar to the H(C)-vcMMAE conjugate and is more potent thanthe H(C)-mcMMAF and H(K)-MCC-DM1 conjugates in this model.

Pharmacokinetics and Toxicokinetics

Mouse pharmacokinetics and rat toxicokinetics were determined fromsingle dose mouse pharmacokinetic and rat toxicology studies (see Tables22 and 23). Mouse pharmacokinetics and rat toxicokinetics weredetermined from single dose mouse pharmacokinetic and rat toxicologystudies. Mouse pharmacokinetics were determined from samples collectedfrom nude mice that were administered a single 3 mg/kg dose. Sampleswere collected for up to 336 h. Rat toxicokinetics were determined inrats (Sprague-Dawley (Crl:CD (SD))) that were administered a singleadministration of H(C)-vc-MMAE or H(C)-#D54 at doses of 3, 10, and 30mg/kg, or administered H(C)-mc-MMAD or H(C)-mc-MMAF at 10, 30, and 100mg/kg. Samples were collected for up to 336 hours. Circulatingconcentrations of total antibody and ADC were measured using ELISAassays. Area under the curve (AUC) was calculated for the total antibodyand ADC for each ADC. ADC to antibody AUC ratios were also calculated.

Exposure of H(C)-#D54 total antibody and ADC were greater than thatobserved for H(C)-vc-MMAE in mice at 3 mg/kg and at all doses evaluatedin rats. The ADC to Ab AUC ratio for H(C)-#D54 was also greater thanthat observed for H(C)-vc-MMAE. These results suggest that H(C)-#D54 hasgreater exposure and that the ADC and/or linker payload are potentiallymore stable than H(C)-vc-MMAE.

Toxicity

The target independent toxicity of #D54 and comparator linker-payloads(mcValCitPABC-MMAD and mcValCitPABC-MMAE) conjugated to a non-crossreactive monoclonal antibody (IgGl) were assessed in a single-dose rattoxicity study with a two-week observation period. The doses of theantibody drug conjugates (ADCs) were 0, 3, 10 and 30 mg/kg with an n is5 males/group and the linker-payload loading was similar among theconjugates (3.8, 3.2 and 4, respectively). These studies included atleast daily clinical observations, weekly body weights, clinicalpathology (end of in-life) and necropsy (Day 15-17) with microscopicexamination of 9 or more tissues and any gross lesions.

Mortality with related body weight changes and signs of morbidity wereobserved at the mg/kg dose for all conjugates and at the 10 mg/kg dosefor the MMAD conjugate. There were no clinical observations or bodyweight changes in the surviving groups.

The target organs of the conjugates identified by microscopicexamination in the surviving dose groups were as follows. The conjugateat 10 mg/kg had debris in the lumen of the epididymis (5/5, minimal tomild), inflammation at the base of the heart (1/5 rats, minimal) andincreased mitosis in the cornea (1/5 rats, minimal). There were nohistological findings for the conjugate at 3 m/kg. For the MMADconjugate in the surviving dose group at 3 mg/kg, there were changes inand related to the bone marrow and in the testis and epididymidis. Forthe MMAE conjugate at 10 mg/kg, there were changes in the bone marrow,kidney, liver and epididymis. At the 3 mg/kg dose for this conjugate,there were kidney changes and increased mitosis in the liver. Thus, instudies of similar design and in the surviving groups, the conjugate didnot have the bone marrow findings seen with the comparator conjugatesand also did not have the liver or kidney findings seen with one of thecomparators.

In summary, the maximum tolerated dose (MTD) of the conjugate and theMMAE conjugate was 10 mg/kg and the MTD of the MMAD conjugate was 3mg/kg. The no observed adverse effect level (NOAEL) of the conjugate was3 mg/kg whereas the NOAEL of the comparator linker-payload conjugateswas less than 3 mg/kg. This study demonstrates how the toxicologicalprofile for #D54 compares to certain compounds described in the art.

Anti-IL-13Rα2 ADC In Vitro and In Vivo Studies

Anti-IL-13Rα2Antibodies and ADCs

The humanized antibody hu08 specifically binds to the IL-13Rα2 receptor.The amino acid and the nucleotide sequences for hu08 are shown in Table3. Kabat CDRs are underlined.

TABLE 3  Amino acid and nucleotide sequences of humanized antibody hu08.SEQ ID NO DESCRIPTION SEQUENCE 9 hu08 heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVR variable regionQAPGKGLEWVATVSSGGSYIYYADSVKGRFTISRDNAK amino acid sequenceNSLYLQMNSLRAEDTAVYYCARQGTTALATRFFDVWG (CDRs underlined). QGTLVTVSS 10hu08 light chain DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQ variable regionKPGKAPKWYSASYRSTGVPSRFSGSGSGTDFTLTISSLQ amino acid sequencePEDFATYYCQHHYSAPWTFGGGTKVEIK (CDRs underlined). 11 hu08 heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFSRNGMSWVR amino acid sequenceQAPGKGLEWVATVSSGGSYIYYADSVKGRFTISRDNAK (CDRs underlined).NSLYLQMNSLRAEDTAVYYCARQGTTALATRFFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSCSPGK 12hu08 light chain DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQamino acid sequence KPGKAPKLLIYSASYRSTGVPSRFSGSGSGTDFTLTISSLQ(CDRs underlined). PEDFATYYCQHHYSAPWTFGGGTKVEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 13hu08-heavy chain GAGGTGCAGCTGGTGGAGTCTGGCGGCGGACTGGTGCnucleotide sequence AGCCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCAGTAGGAATGGCATGTCTTGGGTGAGGCAGGCCCCTGGCAAGGGCCTGGAGTGGGTGGCCACCGTTAGTAGTGGTGGTAGTTACATCTACTATGCAGACAGTGTGAAGGGGCGGTTCACCATCTCCAGGGACAACGCCAAGAACTCCCTGTACCTCCAGATGAACTCCCTGAGGGCCGAGGATACCGCCGTGTACTACTGTGCCAGACAAGGGACTACGGCACTAGCTACGAGGTTCTTCGATGTCTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCTGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA 14hu08-light chain GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCnucleotide sequence CTCTGTGGGCGACAGAGTGACCATCACCTGTAAGGCCAGTCAGGATGTAGGTACTGCTGTAGCCTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTCGGCATCCTACCGGTCCACTGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCCAGCACCATTATAGTGCTCCGTGGACGTTTGGCGGCGGAACAAAGGTGGAGATCAAGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGT

Humanized anti-IL-13Rα2 antibody hu08 was conjugated to variouslinker-payload combinations of the present invention, as provided inTable 4. The antibody drug conjugates were prepared according to themethods of the present invention.

TABLE 4 anti-IL-13Rα2 ADCs. Corresponding ADC ADC Linker-Payload #Nomenclature IL13Ra2-AB08-v1010-hG1-(C)_mcValCitPABC-#54 hu08-vc-0101IL13Ra2-AB08-v1010-hG1-(C)_mc-#115 hu08-mc-3377IL13Ra2-AB08-v1010-hG1-(C)_mc-0#118 hu08-mc-0131IL13Ra2-AB08-v1010-hG1-(C)_MalPeg6C2-#117 hu08-Malpeg-6121IL13Ra2-AB08-v1010-hG1- hu08-Malpeg-0131 (C)_Mal(H2O)Peg6C2-0#118IL13Ra2-AB08-v1010-hG1-(C)_mc-#117 hu08-mc-6121 IL13Ra2-AB08-v1010-hG1-hu08-vc-3906 (C)_mcValCitPABC-#226 IL13Ra2-AB08-v1010-hG1- hu08-vc-6780(C)_mcValCitPABC-#112 IL13Ra2-AB08-v1010-hG1-(C)_mc-#69 hu08-mc-8261IL13Ra2-AB08-v1010-hG1-(C)_mc-#226 hu08-mc-3906IL13Ra2-AB08-v1010-hG1-(C)_MalPeg6C2-#69 hu08-MalPeg-8261huIgG8.84-mcValCitPABC-#54 huIgG8.84-vc0101 huIgG8.84-mc-#115huIgG8.84-mc3377In Vitro Cytotoxicity Assay with Anti-IL-13Rα2ADCs

Cell lines expressing the IL-13Rα2 antigen and a negative control cellline, were cultured with increasing concentrations of anti-IL-13Rα2ADCscomprising the hu08 antibody conjugated to various linker payloads ofthe present invention. After four days, viability of each culture wasassessed. IC₅₀ values were calculated by logistic non-linear regressionand are presented as ng Ab/mL.

The data demonstrates that the anti-IL-13Rα2 antibody huO8v1.0/1.0conjugated to six different auristatin payloads is effective againstboth of the IL-13Rα2 positive cell lines tested (PC3mM2), having an IC₅₀ranging from 1.1 to 4.9 ng Ab/mL or 7.3-32.7 μM (Table 5). All ADCs werenot active against the IL-13Rα2 negative cell line, H460, and thenon-IL-13Rα2 binding control ADCs, huIgG8.84-vc0101 andhuIgG8.84-mc3377, were not active against any of the cell lines tested.

TABLE 5 IC₅₀ (ng Ab/mL) values of humanized anti-IL-13Rα2 ADCs. IC₅₀ (ngAb/mL) ADC DAR PC3MM2 A375 H460 hu08-vc0101 3.2 2.5 3.8 >400000hu08-mc3377 4.3 1.2 2.2 >400000 hu08-mc-0131 3.2 1.3 2.1 >400000hu08-Malpeg-6121 3.3 3.5 3.4 >400000 hu08-Malpeg-0131 2.9 2.94.9 >400000 hu08-mc-6121 3.3 1.1 2.4 >400000 hu08-vc-3906 3 1.52.9 >400000 hu08 vc-6780 4 1.2 2.2 >400000 huIgG8.84-vc01013.7 >400000 >400000 >400000 huIgG8.84-mc3377 4.3 >400000 >400000 >400000In Vivo Subcutaneous Xenograft Models with Anti-IL13Rα2ADCs

The humanized antibody hu08 specifically binds to the IL-13Rα2 receptor.hu08 ADCs with eleven different linker-payload combinations were testedin an in vivo xenograft model. Female, athymic (nude) mice were injectedsubcutaneous with PC3mM2. Mice with staged tumors, approximately 0.1 to0.3 g (n=8 to 10 mice/treatment group), were administered intravenouslyq4d×4 with normal saline (vehicle), huO8v1.0/1.0 ADCs withlinker-payloads vc-0101, vc-6780, vc-3906, mc-8261, mc-0131, mc-6121,mc-3377, MalPeg-8261, MalPeg-0131, MalPeg-6121, or MalPeg-3906, and anon-binding Ab (huIgG8.84) conjugated with vc-0101 or mc-3377, at a doseof 2 or 3 mg Ab/kg. The ADCs were dosed based on Ab content. Tumors weremeasured at least once a week and their size is calculated asmm³=0.5×(tumor width)×(tumor length).

The in vivo efficacy results listed in Table 6 show a range ofanti-tumor activity with the various ADCs tested. The relative order ofpotency ishu08-vc-0101>hu08-vc-6780>hu08-mc-0131>hu08-mc-6121>hu08-mc-3906>hu08-MalPeg-0131>hu08-MalPeg-6121>hu08-MalPeg-3906>>hu08-mc-8261.At the 3 mg/kg dose level, both hu08-vc-0101 and hu08-mc-3377demonstrated antitumor activity, whereas the non-binding Ab (huIgG8.84)conjugated with vc-0101 or mc-3377 had no activity and were similar tothe vehicle control.

TABLE 6 Efficacy of anti-IL-13Rα2 ADCs in PC3MM2 xenografts. Dose PC3MM2xenograft, tumor volume (mm³ +/− SEM) (mg/kg) Day Day Day Day Day DayADC Q4dx4 −1 Day 3 Day 8 16 20 30 42 52 Vehicle 0 638 ± 27 1149 ± 82 1707 ± 133 GT GT GT GT GT hu08- 2 642 ± 36 1036 ± 60  1176 ± 51  GT GTGT GT GT MalPeg- 3906 hu08-mc- 2 642 ± 51 1088 ± 121 1429 ± 158 GT GT GTGT GT 8261 hu08-mc- 2 637 ± 44 1004 ± 73  778 ± 83 GT GT GT GT GT 0131hu08- 2 638 ± 36 947 ± 85 1000 ± 126 GT GT GT GT GT MalPeg- 6121 hu08- 2649 ± 39 1085 ± 54  1040 ± 88  GT GT GT GT GT MalPeg- 0131 hu08-vc- 2646 ± 36 899 ± 54 557 ± 49 243 ± 28 201 ± 20 113 ± 17  207 ± 49  532 ±151 0101 hu08-vc- 2 641 ± 28  850 ± 100 652 ± 54 279 ± 55 217 ± 45 230 ±133 GT GT 6780 hu08-mc- 2 636 ± 37 909 ± 63 821 ± 93 GT GT GT GT GT 6121hu08-mc- 2 637 ± 26 875 ± 48 806 ± 70 GT GT GT GT GT 3906 hu08- 2 645 ±34 991 ± 71 1220 ± 115 GT GT GT GT GT MalPeg- 8261 hu08- 3 339 ± 18 433± 45  38 ± 14  6 ± 6 110 ± 110 230 ± 230 GT GT vc0101 hu08- 3 337 ± 21385 ± 36  41 ± 12  0 ± 0 78 ± 36 346 ± 147 616 ± 243 902 ± 364 mc3377huIgG8.84- 3 365 ± 22 581 ± 47 1017 ± 168 GT GT GT GT GT vc0101huIgG8.84- 10 328 ± 27 459 ± 63  295 ± 121  544 ± 258 GT GT GT GT mc3377GT = group terminated due to large tumor size

Anti-Notch ADC In Vitro and In Vivo Studies

Anti-Notch Antibodies and ADCs

Humanized antibodies, hu28 and hu75, and rat-human chimeric antibodies,ch28 and ch75, specifically bind to the Notch receptor. The amino acidand nucleotide sequences for hu28 and hu75 are provided in Table 7.Kabat CDRs are underlined.

TABLE 7 Amino acid and nucleotide sequences of humanized anti-Notch antibodies.SEQ ID NO. DESCRIPTION SEQUENCES 15 hu28 Heavy ChainEVQLVESGGGLVQPGGSLRLSCAASGFTFRDYGMTWVRQAPGKGL Variable RegionEWVAYISSGSNYIYYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDT amino acid sequenceAVYYCARRGPFVLDAWGQGTLVTVSS (CDRs underlined). 16 hu28 Heavy ChainEVQLVESGGGLVQPGGSLRLSCAASGFTFRDYGMTWVRQAPGKGL amino acid sequenceEWVAYISSGSNYIYYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDT (CDRs underlined).AVYYCARRGPFVLDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG Human IgG1 ConstantGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS RegionVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 17 hu28 Heavy ChainGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGG nucleotide sequenceGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGGGACTATGGAATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCTATATTAGTAGTGGTAGCAATTACATCTATTATGCAGAAGCGGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACGAGGCCCGTTTGTTTTGGATGCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCC CGGGT 18 hu28 Light ChainDIQMTQSPSSLSASVGDRVTITCKASQSINRYLHWYQQKPGKAPKLL Variable RegionIYNANGLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNTWP amino acid sequenceDTFGGGTKVEIK (CDRs underlined). 19 hu28 Light ChainDIQMTQSPSSLSASVGDRVTITCKASQSINRYLHWYQQKPGKAPKLL amino acid sequenceIYNANGLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNTWP (CDRs underlined).DTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE Human kappaAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH Constant RegionKVYACEVTHQGLSSPVTKSFNRGEC 20 hu28 Light ChainGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA nucleotide sequenceGGAGACAGAGTCACCATCACTTGCAAAGCAAGTCAGAGTATTAACAGGTACTTACACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAATGCAAACGGTTTGCAAACGGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTTTGCAGCATAATACGTGGCCGGACACGTTTGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCTCTGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCTCTGCAGTCCGGCAACTCCCAGGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCTCTCCTGTGACCAAGTCC TTCAACCGGGGCGAGTGC 21hu75 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGYAFTDYWMTWVRQAPGKGLVariable Region EWVAEISPNSGGTNFNEKFKGRFTISVDNAKNSLYLQMNSLRAEDTamino acid sequence AVYYCARGEIRYNWFAYWGQGTLVTVSS (CDRs underlined). 22hu75 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGYAFTDYWMTWVRQAPGKGLamino acid sequence EWVAEISPNSGGTNFNEKFKGRFTISVDNAKNSLYLQMNSLRAEDT(CDRs underlined). AVYYCARGEIRYNWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTHuman IgG1 Constant SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSregion LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 23 hu75 Heavy ChainGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGG nucleotide sequenceGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGTTATGCATTCACTGACTACTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCGAAATTTCTCCTAACAGTGGTGGTACTAACTTCAATGAAAAGTTCAAGGGCCGATTCACCATCTCCGTTGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGAAATCCGTTACAATTGGTTTGCTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCCCCGGGT 24hu75 Light Chain DIQMTQSPSSLSASVGDRVTITCKASQNVGNNIAWYQQKPGKAPKLVariable Region LIYYASNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQRLYNSamino acid sequence PFTFGGGTKVEIK (CDRs underlined). 25 hu75 Light ChainDIQMTQSPSSLSASVGDRVTITCKASQNVGNNIAWYQQKPGKAPKL amino acid sequenceLIYYASNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQRLYNS (CDRs underlined).PFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR Human kappaEAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK Constant RegionHKVYACEVTHQGLSSPVTKSFNRGEC 26 hu75 Light ChainGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA nucleotide sequenceGGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGAATGTGGGTAATAATATAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTATGCATCTAACCGGTACACTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATATGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCGTCTTTACAATTCTCCATTCACGTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCCCCTTCCGACGAGCAGCTGAAGTCTGGCACCGCCTCTGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCTCTGCAGTCCGGCAACTCCCAGGAGTCTGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCTCTCCTGTGACCAAGTCCTT CAACCGGGGCGAGTGC

Humanized anti-Notch antibodies, hu28 and hu75, and rat-human chimericanti-Notch antibodies, ch28 and ch75, were conjugated to variouslinker-payload combinations of the present invention, as provided inTable 8. The antibody drug conjugates were prepared according to themethods of the present invention.

TABLE 8 Anti-Notch ADCs. Corresponding ADC ADC Linker-Payload #Nomenclature Notch-28-v1010-hG1-(C)_mcValCitPABC-#54 hu28-vc0101Notch-28-v1010-hG1-(C)_mcValCitPABC-#112 hu28-vc6780Notch-75-v1913-hG1-(C)_mcValCitPABC-#54 hu75-vc0101Notch-75-v1913-hG1-(C)_mcValCitPABC-#112 hu75-vc6780Notch-28-cG1-(C)_mcValCitPABC-#54 ch28-vc0101Notch-28-cG1-(C)_mcValCitPABC-#112 ch28-vc6780 Notch-28-cG1-(C)_mc-#54ch28-mc0101 Notch-28-cG1-(C)_mc-0#118 ch28-mc0131Notch-28-cG1-(C)_mc-#115 ch28-mc3377 Notch-28-cG1-(C)_mc-#69 ch28-mc8261Notch-28-cG1-(C)_MalPeg6C2-0#118 ch28-MalPeg6C2-0131Notch-28-cG1-(C)_MalPeg6C2-#69 ch28-MalPeg6C2-8261Notch-28-cG1-(C)_me-0#118 ch28-me0131 Notch-28-cG1-(C)_m(H2O)c-0#118ch28-m(H2O)c-0131 Notch-75-cG1-(C)_mcValCitPABC-#54 ch75-vc0101Notch-75-cG1-(C)_mcValCitPABC-#112 ch75-vc6780 Notch-75-cG1-(C)_mc-0#118ch75-mc0131 Notch-75-cG1-(C)_mc-#115 ch75-mc3377Notch-75-cG1-(C)_MalPeg6C2-0#118 ch75-MalPegC2-0131Notch-75-cG1-(C)_MalPeg6C2-#69 ch75-MalPeg6C2-8261Notch-75-cG1-(C)_me-0#118 ch75-me0131 Notch-75-cG1-(C)_m(H2O)c-0#118ch75-m(H2O)c-0131 huNeg8.8-(C)_mcValCitPABC-#54 huNeg8.8-vc0101huNeg8.8-(C)_mcValCitPABC-#112 huNeg8.8-vc6780 huNeg8.8-(C)_mc-0#118huNeg8.8-mc0131 huNeg8.8-(C)_mc-#115 huNeg8.8-mc3377huNeg8.8-(C)_me-0#118 huNeg8.8-me0131 huNeg8.8-(C)_MalPeg6C2-#69huNeg8.8-MalPeg6C2- 8261 ch2H6-(C)_mc-#69 ch2H6-mc8261In Vitro Cytotoxicity Assays with Anti-Notch ADCs

The effects of anti-Notch ADCs were assessed on 1) cell linesendogenously expressing Notch protein: HCC2429 (lung cancer), OVCAR3(ovarian cancer) and MDA-MB-468 (breast cancer), 2) cell linesengineered to over-express Notch protein: MDA-MB-468/hNotch andU2OS/hNotch, and 3) a negative control cell line (SW900) using an MTScellular viability indicator (Promega, Madison, Wis.). These cell lineswere cultured with increasing concentrations of anti-Notch ADCscomprising humanized anti-Notch antibodies, hu28 and hu75, and rat-humanchimeric anti-Notch antibodies, ch28 and ch75, conjugated to variouslinker-payload combinations of the present invention. As a specificitycontrol for the anti-Notch-ADCs, non-targeted control-ADCs(huNeg8.8-ADCs or ch2H6-ADCs) were also tested on the same cell lines.After four days, viability of each culture was assessed. IC₅₀ valueswere calculated by logistic non-linear regression and presented as ngAb/mL. The drug antibody ratio (DAR) is also provided.

Table 9 shows IC₅₀ (ng Ab/mL) values of the humanized anti-Notch ADCtreatments. HCC2429 and MDA-MB-468/hNotch cell lines had two individualrepeats. The data demonstrates that the humanized anti-Notch ADCs withvarious linker-payloads were active and induced cell death in the Notchexpressing and over-expressing cancer cell lines HCC2429, OVCAR3,MDA-MB-468, MDA-MB-468/hNotch, U20S/hNotch, but not in the negativecontrol cell line SW900 lacking Notch expression. The non-targetedcontrol-ADCs either lacked potency (LP) and therefore IC₅₀ values werenot generated as indicated, or were minimally active at the highestdoses tested. Anti-Notch ADCs having IC₅₀ values equal to or higher thanIC₅₀ values for control-ADCs were considered to lack potency in vitroand indicted as LP.

TABLE 9 IC₅₀ (ng Ab/mL) values of humanized anti-Notch ADCs. IC₅₀ (ngAb/mL) ± S.E.M. MDA- MDA-MB-468/ U2OS/ ADC DAR HCC2429 OVCAR3 MB-468hNotch hNotch SW900 hu28-vc0101 3.9 473 2940 306 6545 3.2  3 1330 LPhu75-vc0101 3.8 611 3295 515 7001 37 36  523 LP huNeg8.8-vc0101 3.718417 23978 3770 LP 5122 LP LP 23379 hu28-vc6780 3.9 148 2050 17 LP 1.3 3 LP LP hu75-vc6780 4.2 214 630 254 LP 26 25 LP LP huNeg8.8-vc6780 4.2LP LP 9238 LP LP LP LP LP

Table 10 shows IC₅₀ (ng Ab/mL) values of the rat-human chimericanti-Notch ADC treatments. For experiments with 2-4 individual repeats,average IC₅₀ values were calculated along with standard error of themean (S.E.M.). The data demonstrates that the rat-human chimericanti-Notch ADCs with various linker-payloads were active and inducedcell death in the Notch expressing and over-expressing cancer cell linesHCC2429, OVCAR³, MDA-MB-468, MDA-MB-468/hNotch, U20S/hNotch. Thenon-targeted control-ADCs either lacked potency (LP) and therefore IC₅₀values were not generated as indicated, or were minimally active at thehighest doses tested. Anti-Notch ADCs having IC₅₀ values equal to orhigher than IC₅₀ values for control-ADCs were considered to lack potencyin vitro and indicted as LP.

TABLE 10 IC₅₀ (ng Ab/mL) values of rat-human chimeric anti-Notch ADCs(nd = not determined). IC₅₀ (ng Ab/mL) ± S.E.M. MDA-MB-468/ U2OS/ ADCDAR HCC2429 OVCAR3 MDA-MB-468 hNotch hNotch ch28-mc8261 3.7 LP nd 12147± 4806.4  nd nd ch2H6-mc8261 4.1 LP nd LP nd nd ch28-MalPeg6C2-8261 4.3LP nd 83 ± 35.5 nd nd ch75-MalPeg6C2-8261 3.8 LP nd 4255 ± 2375   nd ndhuNeg8.8-MalPeg6C2-8261 4.1 LP nd LP nd nd ch28-mc0131 3.4 251 ± 77.5 6± 1.0 35 ± 18.5 nd 3 ± 0.5  ch75-mc0131 3.3  671 ± 406.5 289 8202 ±2773.0 nd 19 huNeg8.8-mc0131 3.9 Nd LP LP nd LP ch28-me0131 3.9 30 8 ±2.0 24 ± 14.0 nd 3 ± 1.15 ch75-me0131 3.5 Nd nd 259 nd ndhuNeg8.8-me0131 3.7 Nd LP LP nd LP ch28-mc3377 3.7 LP 14 ± 5.5  27 ±11.3 nd 3 ± 0.5  ch75-mc3377 3.7 Nd nd 560 nd nd huNeg8.8-mc3377 3.6 NdLP LP nd LP ch28-MalPeg6C2-0131 4.1 LP 10 ± 2.0  10 ± 1.0  nd 3 ± 0.85ch28-vc0101 3.8  3230 ± 1116.5 635 5443 ± 2630.9 4 ± 0.5 95 ± 18.2 ch75-vc0101 2.7 2112 ± 826.0 LP 4064 ± 1793.9 24 ± 4.0  LPhuNeg8.8-vc0101 3.7 15341 LP 4523 ± 2789.5 8833 LP ch28-vc6780 4.1 324 ±78.9 90 ± 48.5 4407 ± 2128.2 3 ± 0.5 LP ch75-vc6780 2.8 1004 ± 177.0 9226873 ± 4230.0 21 ± 3.5  LP huNeg8.8-vc6780 4.1 LP LP LP LP LPIn vivo Human Tumor Xenograft Models with Anti-Notch ADCs

Humanized anti-Notch antibodies, hu28 and hu75, and rat-human chimericanti-Notch antibodies, ch28 and ch75, were conjugated to variouslinker-payload combinations and tested in 37622A1 non-small cell lungcancer (NSCLC), HCC2429 lung cancer, MDA-MB-468 breast cancer and N87gastric cancer xenograft models. For each model described below thefirst dose was given on Day 0. The tumors were measured at least once aweek and their volume was calculated with the formula: tumor volume(mm³)=0.5×(tumor width²)(tumor length). The mean tumor volumes (±S.E.M.)for each treatment group were calculated having a maximum of 10 animalsand a minimum of 6 animals to be included.

A. 37622A1 NSCLC Xenografts

The effects of anti-Notch ADCs were examined in immunodeficient mice onthe in vivo growth of human tumor xenografts that were established fromfragments of freshly resected 37622A1 NSCLC tumors obtained inaccordance with appropriate consent procedures (Asterand). The 37622A1NSCLC patient-derived xenografts were subcutaneously passaged in vivo asfragments from animal to animal in nude (Nu/Nu) female mice. When thetumors reached a volume of 150 to 300 mm³, they were staged to ensureuniformity of the tumor size among various treatment groups. The 37622A1NSCLC model was dosed intraveneously four times every four days (Q4dx4)with PBS vehicle, humanized anti-Notch ADCs, control huNeg-8.8 ADCs andcisplatin at the doses provided in Table 11.

Cisplatin is a platinum-based anti-cancer agent used in the treatment ofcancer and considered a standard-of-care therapy. Cisplatin cross-linksDNA thereby inducing apoptosis and cell growth inhibition. The datademonstrates that anti-Notch ADCs hu28-vc0101, hu28-vc6780, hu75-vc0101and hu75-vc6780 inhibited growth of 37622A1 NSCLC xenografts. Further,the data shows that anti-Notch ADCs inhibited tumor growth more potentlythan control huNeg8.8-ADCs. Furthermore, the data shows that anti-NotchADCs inhibited tumor growth more potently than cisplatin indicating agreater potency than a platinum-based standard-of-care chemotherapeuticdrug.

TABLE 11 Efficacy of anti-Notch ADCs in 37622A1 NSCLC xenografts.37622A1 NSCLC xenografts, tumor volume (mm³ ± SEM) hu28- hu28- hu75-hu75- huNeg-8.8- huNeg-8.8- PBS vc0101 vc6780 vc0101 vc6780 vc0101vc6780 Cisplatin Dose mg/kg 0 3 10 3 10 3 10 5 DAY −1 187 ± 10 186 ± 13182 ± 16 185 ± 17  183 ± 17 184 ± 18 182 ± 17 185 ± 11 DAY 4 227 ± 19202 ± 16 176 ± 13 200 ± 16  205 ± 23 225 ± 17 226 ± 26 226 ± 15 DAY 7279 ± 24 202 ± 15 176 ± 19 227 ± 16  195 ± 22 274 ± 18 265 ± 28 280 ± 29DAY 11 371 ± 42 130 ± 11 122 ± 10 175 ± 20  147 ± 23 309 ± 26 246 ± 30301 ± 34 DAY 14 419 ± 49 119 ± 11 95 ± 7 156 ± 19  118 ± 18 303 ± 26 277± 41 345 ± 47 DAY 18 516 ± 63 71 ± 6 65 ± 6 112 ± 16   93 ± 14 298 ± 28219 ± 31 309 ± 37 DAY 21 562 ± 65 55 ± 6 56 ± 6 122 ± 27   98 ± 20 320 ±41 218 ± 42 373 ± 50 DAY 25 610 ± 78 49 ± 6 51 ± 6 137 ± 33   93 ± 24315 ± 52 264 ± 52 401 ± 58 DAY 28 624 ± 94 41 ± 7 51 ± 8 161 ± 53   99 ±26 358 ± 61 246 ± 51 446 ± 64 DAY 32 817 ± 99  42 ± 13  72 ± 15 175 ±52  165 ± 45 398 ± 64 332 ± 77 482 ± 62 DAY 35  900 ± 104  42 ± 11  92 ±21 271 ± 79  229 ± 59 487 ± 79 384 ± 94 587 ± 80 DAY 39  960 ± 117  62 ±26 120 ± 31 319 ± 103 294 ± 78  569 ± 102  431 ± 114 591 ± 83 DAY 42 931 ± 108  75 ± 34 151 ± 37 357 ± 113 318 ± 71  590 ± 101  495 ± 128612 ± 92 DAY 46 1037  92 ± 44 172 ± 47 431 ± 137  412 ± 106  743 ± 133 610 ± 165  723 ± 119  102 DAY 49 1119 ± 120 120 ± 63 248 ± 62 519 ± 135 521 ± 132  810 ± 121  718 ± 202  853 ± 139 DAY 53 1345 ± 158 144 ± 67339 ± 93 678 ± 195  629 ± 162  989 ± 146  848 ± 251  970 ± 193 DAY 561485 ± 185 126 ± 51  376 ± 100 818 ± 251  808 ± 196 1149 ± 191  776 ±184 1215 ± 231 DAY 60 1691 ± 220 180 ± 85  503 ± 138 710 ± 162  917 ±209 1287 ± 194  964 ± 232 1428 ± 273 DAY 63 1736 ± 193  223 ± 111  604 ±160 824 ± 191  917 ± 147 1503 ± 227 1097 ± 254 — DAY 67 —  296 ± 152 888 ± 272 938 ± 202 1116 ± 173 1600 ± 251 1167 ± 260 — DAY 70 —  312 ±162  773 ± 235 953 ± 209 1181 ± 203 — 1352 ± 305 — DAY 74 —  331 ± 160 881 ± 264 — — — — — DAY 77 —  422 ± 210 1029 ± 325 — — — — — DAY 81 — 510 ± 248 — — — — — — DAY 84 —  622 ± 322 — — — — — —B. HCC2429 Lung Xenografts

Similar in vivo experiments were performed with the HCC2429 lung cancercell line as described above. To generate xenografts, nude (Nu/Nu)female mice were implanted subcutaneously with 3.5×10⁶ HCC2429 cells in50% Matrigel (BD Biosciences). When the tumors reached a volume of 200to 400 mm³, the tumors were staged to ensure uniformity of the tumormass among various treatment groups. The HCC2429 lung model was dosedintraveneously Q4dx4 with PBS vehicle, humanized anti-Notch ADCs andcontrol huNeg-8.8 ADCs at the doses provided in Tables 12 and 13. Thedata demonstrates that anti-Notch ADCs hu28-vc0101, hu28-vc6780,hu75-vc0101 and hu75-vc6780 inhibited growth of HCC2429 lung xenograftsin a dose-dependent manner. Further, the data shows that anti-Notch ADCsinhibited tumor growth more potently than control huNeg8.8-ADCs at the 1and 3 mg/kg doses for anti-Notch ADCs with vc0101 linker-payloads and atthe 3 and 10 mg/kg doses for anti-Notch ADCs with vc6780linker-payloads. Furthermore, the data demonstrates that a 3 mg/kg doseof hu28-vc0101 was more potent than a 10 mg/kg dose of hu28-vc6780.

TABLE 12 Efficacy of anti-Notch-vc0101 ADCs in HCC2429 lung xenografts.HCC2429 Lung xenografts, tumor volume (mm³ +/− SEM) PBS hu28-vc0101hu75-vc0101 huNeg-8.8-vc0101 Dose mg/kg 0 3 1 0.3 3 1 0.3 3 1 0.3 DAY −1245 ± 24 245 ± 23  246 ± 26 246 ± 30 245 ± 28  246 ± 23 247 ± 29 244 ±30 245 ± 33 246 ± 27 DAY 1 529 ± 52 548 ± 52  532 ± 36 528 ± 50 498 ±39  548 ± 37 524 ± 66 482 ± 59 519 ± 72 514 ± 50 DAY 3 742 ± 73 606 ±78  757 ± 68 733 ± 78 498 ± 44  753 ± 93 713 ± 74 695 ± 91 756 ± 97 724± 73 DAY 6 1205 ± 120 723 ± 101 1095 ± 119 1112 ± 132 469 ± 70  1096 ±146 1078 ± 74  1075 ± 132 1144 ± 100 1207 ± 100 DAY 8 1720 ± 181 696 ±100 1324 ± 173 1617 ± 172 407 ± 71  1428 ± 200 1499 ± 115 1404 ± 1831598 ± 133 1683 ± 165 DAY 10 2312 ± 197 620 ± 90  1606 ± 250 2027 ± 233370 ± 81  1611 ± 189 1830 ± 120 1735 ± 253 1974 ± 185 2163 ± 260 DAY 133235 ± 120 543 ± 92  1717 ± 223 2642 ± 297 273 ± 69  1803 ± 208 2408 ±226 2162 ± 376 2676 ± 346 2589 ± 287 DAY 15 — 512 ± 111 1865 ± 263 — 298± 88  1871 ± 232 — — — — DAY 17 — 442 ± 114 2228 ± 333 — 250 ± 77  1948± 228 — — — — DAY 20 — 428 ± 144 — — 177 ± 44  — — — — — DAY 23 — 405 ±149 — — 160 ± 35  — — — — — DAY 27 — 422 ± 164 — — 174 ± 51  — — — — —DAY 30 — 394 ± 182 — — 196 ± 72  — — — — — DAY 34 — 505 ± 236 — — 295 ±121 — — — — — DAY 37 — 606 ± 283 — — 433 ± 179 — — — — — DAY 41 — 750 ±361 — — 606 ± 259 — — — — — DAY 45 — 872 ± 415 — — 836 ± 359 — — — — —DAY 49 — 558 ± 303 — — 732 ± 350 — — — — — DAY 52 — 571 ± 310 — — — — —— — — DAY 56 — 704 ± 399 — — — — — — — —

TABLE 13 Efficacy of anti-Notch-vc6780 ADCs in HCC2429 lung xenografts.HCC2429 Lung xenografts, tumor volume (mm³ ± SEM) PBS hu28-vc6780hu75-vc6780 huNeg-8.8-vc6780 Dose mg/kg 0 10 3 1 10 3 1 10 3 1 DAY 245 ±28 244 ± 22 245 ± 24 245 ± 27 244 ± 19 246 ± 30 245 ± 16 244 ± 22 244 ±26 245 ± 20 −1 DAY 398 ± 50 369 ± 31 379 ± 45 400 ± 66 407 ± 43 403 ± 51418 ± 34 429 ± 56 427 ± 49 402 ± 53 1 DAY  701 ± 102 318 ± 31 493 ± 65 579 ± 113 339 ± 36 526 ± 74 629 ± 65 619 ± 62 689 ± 83 655 ± 97 3 DAY 949 ± 140 228 ± 28 609 ± 82  826 ± 191 251 ± 33 615 ± 98 916 ± 97  808± 101  965 ± 114  837 ± 117 5 DAY 1345 ± 200 172 ± 22 638 ± 86 1023 ±259 225 ± 24  615 ± 115 1164 ± 131 1072 ± 154 1380 ± 136 1099 ± 172 7DAY 2045 ± 356 143 ± 22  784 ± 115 1439 ± 398 198 ± 24  717 ± 129 1705 ±184 1452 ± 210 2082 ± 192 1722 ± 363 10 DAY — 134 ± 20  883 ± 132 1442 ±487 166 ± 22  807 ± 130 2029 ± 270 1673 ± 290 2701 ± 228 1586 ± 337 12DAY — 115 ± 16  895 ± 175 — 150 ± 22  831 ± 145 2294 ± 287 1809 ± 314 —— 14 DAY — 127 ± 18 1105 ± 253 — 158 ± 32 1017 ± 178 — — — — 17 DAY —149 ± 27 1219 ± 311 — 164 ± 48 1297 ± 231 — — — — 20 DAY — 206 ± 60 1618± 468 — 261 ± 89 1813 ± 343 — — — — 24 DAY —  290 ± 100 — —  316 ± 1351970 ± 462 — — — — 27 DAY —  378 ± 150 — —  438 ± 201 — — — — — 31 DAY — 551 ± 244 — —  423 ± 177 — — — — — 34 DAY —  718 ± 332 — —  504 ± 203 —— — — — 38 DAY — 1011 ± 504 — —  655 ± 266 — — — — — 42 DAY — — — —  793± 320 — — — — — 46 DAY — — — —  901 ± 351 — — — — — 49 DAY — — — — 1228± 472 — — — — — 53

The HCC2429 lung model was also dosed intravenously Q4dx4 with PBSvehicle, rat-human chimeric anti-Notch ADCs and control huNeg-8.8 ADCs,at a dose of 5 mg/kg as provided in FIG. 8A. The data demonstrates thatanti-Notch ADCs with non-cleavable (mc) and cleavable (vc) linkers andvarious payload combinations inhibited growth of HCC2429 lungxenografts. Further, the data shows that rat-human chimeric anti-NotchADCs inhibited tumor growth more potently than control huNeg8.8-ADCs.Furthermore, the data demonstrates that rat-human chimeric anti-NotchADCs with vc0101 linker-payloads were more potent than the otheranti-Notch ADCs tested.

C. MDA-MB-468 Breast Xenografts

Similar in vivo experiments were performed with the MDA-MB-468 breastcancer cell line as described above. MDA-MB-468 cells are classified asa triple-negative breast cancer (TNBC) basal-like subtype since theylack expression of the estrogen receptor, progesterone receptor andhuman epidermal growth factor receptor 2 (HER²) (Lehmann, B D, et al, JClin Invest. 2011; 121(7):2750-2767). To generate xenografts, femaleSCID Hairless Outbred (SHO) mice were orthotopically implanted with10×10⁶ MDA-MB-468 cells containing 50% Matrigel (BD Biosciences) in themammary fat pad. When the tumors reached a volume of 250 to 450 mm³, thetumors were staged to ensure uniformity of the tumor mass among varioustreatment groups. The MDA-MB-468 breast model was dosed intraveneouslyQ4dx4 with PBS vehicle, humanized anti-Notch ADCs and control huNeg-8.8ADCs at the doses provided in Tables 14 and 15. The data demonstratesthat anti-Notch ADCs hu28-vc0101, hu28-vc6780, hu75-vc0101 andhu75-vc6780 inhibited growth of MDA-MB-468 breast xenografts in adose-dependent manner. Further, the data shows that anti-Notch ADCsinhibited tumor growth more potently than control huNeg8.8-ADCs at the 1and 3 mg/kg doses for ADCs with vc0101 linker-payloads and 1, 3 and 10mg/kg doses for ADC with vc6780 linker-payloads. Furthermore, the datademonstrates that a 1 mg/kg dose of anti-Notch ADCs with vc0101linker-payloads were more potent than a 3 mg/kg dose of anti-Notch ADCswith vc6780 linker-payloads.

TABLE 14 Efficacy of anti-Notch-vc0101 ADCs in MDA-MB-468 breastxenografts. MDA-MB-468 Breast xenografts, tumor volume (mm³ ± SEM) PBShu28-vc0101 hu75-vc0101 huNeg-8.8-vc0101 Dose mg/kg 0 3 1 0.3 3 1 0.3 31 0.3 DAY 0 343 ± 12 347 ± 15  348 ± 22  336 ± 19 347 ± 20  347 ± 22 348± 21 334 ± 23 346 ± 16 344 ± 19 DAY 4 441 ± 24 359 ± 24  439 ± 21  403 ±28 444 ± 28  410 ± 32 439 ± 31 424 ± 29 447 ± 32 442 ± 19 DAY 7 469 ± 32326 ± 27  415 ± 25  395 ± 38 338 ± 20  383 ± 33 435 ± 27 411 ± 26 449 ±20 438 ± 23 DAY 11 495 ± 28 227 ± 27  372 ± 34  412 ± 42 277 ± 22  373 ±33 504 ± 38 439 ± 36 538 ± 23 496 ± 37 DAY 14 581 ± 35 147 ± 20  314 ±27  488 ± 45 181 ± 19  350 ± 40 507 ± 30 445 ± 29 592 ± 47 560 ± 36 DAY18 639 ± 43 77 ± 10 261 ± 33  497 ± 55 90 ± 12 296 ± 33 587 ± 44 479 ±42 619 ± 42 578 ± 36 DAY 21 638 ± 46 16 ± 8  219 ± 41  509 ± 60 60 ± 9 260 ± 49 590 ± 55 481 ± 34 676 ± 46 627 ± 30 DAY 26 707 ± 41 0 ± 0 253 ±61  590 ± 66 16 ± 10 267 ± 59 652 ± 64 548 ± 41 793 ± 54 671 ± 56 DAY 29749 ± 59 0 ± 0 238 ± 64  — 8 ± 8 261 ± 62 675 ± 63 — 819 ± 73 669 ± 37DAY 32 812 ± 80 0 ± 0 266 ± 67  — 7 ± 7 264 ± 67 738 ± 70 — 913 ± 72 758± 44 DAY 35 891 ± 79 0 ± 0 271 ± 73  — 0 ± 0 326 ± 86 821 ± 69 — 1023 ±96  848 ± 58 DAY 39 892 ± 84 0 ± 0 310 ± 88  — 0 ± 0 324 ± 81 864 ± 74 —— 884 ± 64 DAY 42 1037 ± 104 0 ± 0 349 ± 95  — 0 ± 0 381 ± 94 997 ± 84 —— 1002 ± 55  DAY 47 1173 ± 134 0 ± 0 394 ± 123 — 0 ± 0 442 ± 69 — — —1145 ± 78  DAY 50 — 0 ± 0 377 ± 118 — 0 ± 0 484 ± 89 — — — 1120 ± 67 DAY 53 — 0 ± 0 414 ± 127 — 0 ± 0 452 ± 78 — — — 1229 ± 100 DAY 56 — 0 ±0 470 ± 128 — 0 ± 0 535 ± 93 — — — 1314 ± 120 DAY 60 — 0 ± 0 532 ± 140 —0 ± 0 603 ± 98 — — — — DAY 63 — 0 ± 0 509 ± 117 — 0 ± 0 — — — — — DAY 67— 0 ± 0 611 ± 148 — 0 ± 0 — — — — — DAY 70 — 0 ± 0 — — 0 ± 0 — — — — —

TABLE 15 Efficacy of anti-Notch-vc6780 ADCs in MDA-MB-468 breastxenografts. MDA-MB-468 Breast xenografts, tumor volume (mm³ ± SEM) PBShu28-vc6780 hu75-vc6780 huNeg-8.8-vc6780 Dose mg/kg 0 10 3 1 10 3 1 10 31 DAY 0 342 ± 9  335 ± 9  342 ± 18 342 ± 16 343 ± 10 344 ± 11 340 ± 14339 ± 18  341 ± 12 346 ± 16 DAY 4 466 ± 20 395 ± 19  394 ± 33 462 ± 22418 ± 15 406 ± 22 423 ± 27 432 ± 45  457 ± 23 466 ± 29 DAY 7 481 ± 17350 ± 19  399 ± 24 452 ± 30 370 ± 18 378 ± 21 434 ± 29 449 ± 45  529 ±24 528 ± 25 DAY 11 611 ± 44 248 ± 26  380 ± 25 512 ± 35 302 ± 21 403 ±21 471 ± 39 504 ± 38  599 ± 23 621 ± 43 DAY 14 610 ± 19 154 ± 23  401 ±30 507 ± 38 228 ± 19 370 ± 28 470 ± 44 503 ± 64  622 ± 31 639 ± 48 DAY19 707 ± 34 65 ± 17 438 ± 39 538 ± 47 112 ± 23 339 ± 19 536 ± 49 437 ±54  697 ± 36 713 ± 48 DAY 22 — 25 ± 16 414 ± 41 551 ± 48  52 ± 21 360 ±17 552 ± 44 415 ± 54  — — DAY 25 — 26 ± 19 491 ± 37 575 ± 55  63 ± 25381 ± 23 597 ± 48 421 ± 76  — — DAY 28 — 15 ± 15 497 ± 68 654 ± 74  64 ±26 443 ± 33 660 ± 53 451 ± 84  — — DAY 32 0 ± 0 524 ± 69 653 ± 82  71 ±31 437 ± 28 634 ± 74 456 ± 94  DAY 35 — 0 ± 0 — 734 ± 89  85 ± 38 495 ±33 742 ± 80 541 ± 108 — — DAY 40 — 0 ± 0 — 761 ± 99 125 ± 44 535 ± 41794 ± 87 563 ± 109 — — DAY 43 — 0 ± 0 —  816 ± 122 134 ± 42 619 ± 47 878± 78 581 ± 120 — — DAY 46 — 0 ± 0 —  859 ± 126 143 ± 42 636 ± 38 868 ±99 617 ± 116 — — DAY 49 — 0 ± 0 —  948 ± 178 159 ± 44 723 ± 71  996 ±109 733 ± 129 — — DAY 53 — 0 ± 0 — 1008 ± 192 201 ± 63 795 ± 67 — 758 ±163 — — DAY 56 — 0 ± 0 — — 211 ± 63 819 ± 77 — — — — DAY 60 — 0 ± 0 — —240 ± 63  976 ± 115 — — — — DAY 63 — 0 ± 0 — — 201 ± 57 — — — — —

The MDA-MB-468 breast model was also dosed intraveneously Q4dx4 with PBSvehicle, rat-human chimeric anti-Notch ADCs and control huNeg-8.8 ADCs,at a dose of 5 mg/kg as provided in FIGS. 8B and 8C. The datademonstrates that rat-human chimeric anti-Notch ADCs with non-cleavable(mc) and cleavable (vc) linkers and various payload combinationsinhibited growth of MDA-MB-468 breast xenografts. Further, the datashows that rat-human chimeric anti-Notch ADCs inhibited tumor growthmore potently than control huNeg8.8-ADCs. Furthermore, the datademonstrates that rat-human chimeric anti-Notch ADCs with vc0101linker-payloads were more potent than the other rat-human chimericanti-Notch ADCs tested.

D. N87 Gastric Xenografts

Similar in vivo experiments were performed with the N87 gastric cancercell line as described above. To generate xenografts, nude (Nu/Nu)female mice were implanted subcutaneously with 7.5×10⁶ N87 cells in 50%Matrigel (BD Biosciences). When the tumors reached a volume of 250 to450 mm³, the tumors were staged to ensure uniformity of the tumor massamong various treatment groups. The N87 gastric model was dosedintraveneously Q4dx4 with PBS vehicle, humanized anti-Notch ADCs,control huNeg-8.8 ADCs and cisplatin at the doses provided in Tables 16and 17. The data demonstrates that anti-Notch ADCs hu28-vc0101,hu28-vc6780, hu75-vc0101 and hu75-vc6780 inhibited growth of N87 gastricxenografts in a dose-dependent manner. Further, the data shows thatanti-Notch ADCs inhibited tumor growth more potently than controlhuNeg8.8-ADCs at the 1, 3, 5 mg/kg doses for ADCs with vc0101linker-payloads and 3 and 10 mg/kg doses for ADCs with vc6780linker-payloads. Furthermore, the data demonstrates that ADCs withvc0101 linker-payloads were in general more potent than cisplatinstandard-of-care therapy and ADCs with vc6780 linker-payloads.

TABLE 16 Efficacy of anti-Notch-vc0101 ADCs in N87 gastric xenografts.N87 Gastric xenografts, tumor volume (mm³ ± SEM) PBS hu28-vc0101hu75-vc0101 huNeg-8.8-vc0101 Cisplatin Dose mg/kg 0 1 3 5 1 3 5 1 3 5 5DAY 0 327 ± 11 321 ± 21  326 ± 13 321 ± 8  321 ± 9  324 ± 19 320 ± 11 327 ± 18  324 ± 11 321 ± 16  328 ± 20 DAY 4 526 ± 19 369 ± 18  339 ± 11344 ± 14  392 ± 15  362 ± 35 315 ± 15  437 ± 34  478 ± 19 423 ± 32  414± 27 DAY 7 706 ± 27 429 ± 43  302 ± 10 272 ± 7  417 ± 25  303 ± 21 246 ±12  584 ± 54  625 ± 34 512 ± 34  520 ± 26 DAY 11 854 ± 36 304 ± 30  182± 14 152 ± 13  331 ± 21  174 ± 14 156 ± 10  702 ± 60  716 ± 53 501 ± 38 501 ± 29 DAY 14 887 ± 45 282 ± 25  191 ± 5  155 ± 13  305 ± 17  172 ± 10151 ± 7  822 ± 65  823 ± 42 549 ± 37  637 ± 31 DAY 18 1045 ± 68  263 ±24  161 ± 7  138 ± 11  267 ± 17  151 ± 10 128 ± 6  823 ± 73  789 ± 33491 ± 51  — DAY 21 1072 ± 76  227 ± 23  123 ± 15 110 ± 9  218 ± 23  130± 5  115 ± 7  857 ± 78  785 ± 35 413 ± 50  — DAY 26 1303 ± 140 205 ± 32 108 ± 16 69 ± 16 185 ± 24   92 ± 14 82 ± 10 895 ± 126 825 ± 62 343 ± 63 — DAY 29 1276 ± 139 180 ± 30   99 ± 14 50 ± 13 211 ± 37  104 ± 16 75 ±12 957 ± 126 879 ± 72 411 ± 89  — DAY 33 1480 ± 183 211 ± 43  106 ± 1743 ± 14 251 ± 53   91 ± 18 73 ± 12 988 ± 180 966 ± 98 411 ± 89  — DAY 36— 215 ± 42  122 ± 22 52 ± 16 272 ± 59   86 ± 18 85 ± 9  884 ± 143 1023 ±106 481 ± 86  — DAY 39 — 261 ± 54  128 ± 23 45 ± 14 304 ± 72   59 ± 1672 ± 13 937 ± 167 1142 ± 121 535 ± 128 — DAY 42 — 283 ± 52  149 ± 25 34± 15 314 ± 73   81 ± 22 74 ± 13 1008 ± 179  1240 ± 143 596 ± 119 — DAY47 — 262 ± 64  105 ± 19 25 ± 14 334 ± 95   80 ± 25 36 ± 8  1061 ± 210 1380 ± 153 621 ± 137 — DAY 53 — 302 ± 75  104 ± 30 29 ± 16 393 ± 115  86± 24 69 ± 13 — — 757 ± 189 — DAY 62 — 415 ± 111 116 ± 47 33 ± 18 463 ±155 106 ± 35 50 ± 15 — — 690 ± 122 — DAY 70 — 521 ± 135 139 ± 54 58 ± 30658 ± 241 148 ± 54 76 ± 22 — — 852 ± 150 —

TABLE 17 Efficacy of anti-Notch-vc6780 ADCs in N87 gastric xenografts.N87 Gastric xenografts, tumor volume (mm³ ± SEM) PBS hu28-vc6780hu75-vc6780 huNeg8.8-vc6780 Dose mg/kg 0 10 3 1 10 3 1 10 3 DAY 0 345 ±14 350 ± 14 349 ± 10 348 ± 13 349 ± 8  351 ± 20 359 ± 16 356 ± 20  344 ±14 DAY 4 600 ± 16 434 ± 24 552 ± 24 560 ± 26 468 ± 18 545 ± 37 552 ± 40581 ± 60  537 ± 36 DAY 8 675 ± 20 379 ± 12 545 ± 37 592 ± 44 351 ± 24511 ± 31 568 ± 62 605 ± 67  670 ± 45 DAY 11 763 ± 54 315 ± 18 511 ± 25617 ± 48 316 ± 25 544 ± 43 591 ± 63 636 ± 79  706 ± 38 DAY 14 886 ± 72292 ± 24 564 ± 29 782 ± 60 269 ± 27 558 ± 36 666 ± 77 775 ± 117 917 ± 36DAY 18 997 ± 93 199 ± 18 479 ± 29 797 ± 88 224 ± 26 494 ± 41 642 ± 90665 ± 112 958 ± 57 DAY 21 1041 ± 107 194 ± 20 499 ± 34 839 ± 93 192 ± 19534 ± 41  710 ± 117 637 ± 119 1002 ± 59  DAY 25 1151 ± 144 181 ± 21 588± 40  878 ± 105 227 ± 32 628 ± 58  748 ± 138 647 ± 134 1075 ± 82  DAY 281200 ± 155 204 ± 16 672 ± 48  904 ± 123 244 ± 35 645 ± 57  763 ± 145 674± 146 1148 ± 77  DAY 33 1481 ± 206 196 ± 27 786 ± 65 1043 ± 152 267 ± 52730 ± 66  991 ± 239 733 ± 195 1290 ± 128 DAY 36 — 189 ± 37 827 ± 69 1108± 185 300 ± 64 850 ± 74 — 817 ± 222 1265 ± 111 DAY 39 — 228 ± 44 847 ±77 1204 ± 209 323 ± 69 881 ± 88 — 880 ± 247 1429 ± 121 DAY 42 — 257 ± 60959 ± 81 — 350 ± 78 1020 ± 99  — 797 ± 244 — DAY 46 — 253 ± 59 1018 ±94  — 380 ± 78 1097 ± 129 — 874 ± 267 — DAY 50 — 253 ± 67 1111 ± 95  —415 ± 77 1162 ± 134 — — — DAY 56 — 298 ± 85 1279 ± 108 —  504 ± 111 1331± 187 — — — DAY 63 — 345 ± 93 1368 ± 133 —  581 ± 121 — — — — DAY 70 — 376 ± 117 1483 ± 154 —  726 ± 163 — — — — DAY 77 —  388 ± 123 — —  797± 184 — — — —

The N87 gastric model was also dosed intraveneously Q4dx4 with PBSvehicle, rat-human chimeric anti-Notch ADCs and control huNeg-8.8 ADCs,at a dose of 5 mg/kg as provided in FIG. 8D. The data demonstrates thatrat-human chimeric anti-Notch ADCs with non-cleavable (mc) and cleavable(vc) linkers and various payload combinations inhibited growth of N87gastric xenografts. Further, the data shows that rat-human chimericanti-Notch ADCs inhibited tumor growth more potently than controlhuNeg8.8-ADCs. Furthermore, the data demonstrates that rat-humanchimeric anti-Notch ADCs with vc0101 linker-payloads were more potentthan the other anti-Notch ADCs tested.

The N87 gastric model was also dosed intravenously Q4dx4 with PBSvehicle and rat-human chimeric anti-Notch ADCs ch28-mc0131, ch75-mc0131,ch28-m(H2O)c-0131 and ch75-m(H2O)c-0131 at a dose of 5 mg/kg as providedin FIG. 8E. The data demonstrates that rat-human chimeric anti-NotchADCs having mc0131 and m(H2O)c-0131 linker-payloads inhibited growth ofN87 gastric xenografts. Further, the data demonstrates that rat-humanchimeric anti-Notch ADCs having m(H2O)c-0131 linker-payloads were morepotent than rat-human chimeric anti-Notch ADCs having mc0131linker-payloads.

TABLE 18A Selected compounds (cytotoxic peptides with linkers) of theinvention Quantity Preparation Purification in mg Linker-Payload #method method (Yield) mcValCitPABC-#34 General Method D 4.7 (12%)procedure E MalPeg3C2-#41 General Method C 36 (28%) procedure DMalPeg6C2-#42 General Method C 125 (88%) procedure D mc-#44 GeneralMethod C 5.9 (25%) procedure D MalPeg3C2-#44 General Method C 10 (28%)procedure D MalPeg6C2-#44 General Method C 1.8 (6%) procedure DMalValCitPABC-#44 General Method F 4 (10%) procedure E mc-#45 GeneralMethod C 12.8 (53%) procedure D MalPeg3C2-#45 General Method C 7.5 (28%)procedure D MalPeg6C2-#45 General Method E1* 13.6 (45%) procedure DmcValCitPABC-#45 General Method C & 5.8 (15%) procedure E Method E1mcValCitPABC-#54 General Method D 33 (36%) procedure E mc-#69 GeneralMethod C 30.2 (24%) procedure D MalPeg6C2-#69 General Method C 3.6 (13%)procedure D mcValCitPABC-#69 General Method I 51 (9%) procedure EmcValCitPABC-#70 General Method D 6.9 (12%) procedure E mcValCitPABC-#75General Method H 5.3 (14%) procedure E mc-#79 General Method E 5.6 (19%)procedure D mcValCitPABC-#79 General Method D 5.5 (10%) procedure EmcValCitPABC-#92 General Method E 9.5 (26%) procedure EmcValCitPABC-#112 General Method C 11.8 (21%) procedure E mv-#115General Method J 1.4 (3.3%) procedure D mc-#115 — Method L 124 (11%)mb-#115 General — — procedure D me-#115 General — — procedure DmcValCitPABC-#115 General Method K 4.9 (12%) procedure E mc-#51 GeneralMethod E1 5.5 (42%) procedure D mc-#47 General Method E1 8.4 (46%)procedure D mc-#54 General Method E1 12.9 (65%) procedure DmcValCitPABC-#47 General Method E1 3.3 (20%) procedure EmcValCitPABC-#26 General Method E1 2.3 (20%) procedure E mc-#26 GeneralMethod E1 5.4 (10%) procedure D mcValCitPABC-#42 General Method E1 10.8(38%) procedure E mcValCitPABC-#36 General Method E1 12.6 (32%)procedure E mc-#42 General Method E1 7.1 (83%) procedure D AmPeg6C2-#54General Method J 44 (67%) procedure N MalPeg3C2-#54 General Method J 19(69%) procedure D mcValCitPABCAmPeg6C2-#54 General Method J 12 (42%)procedure O mcValCitPABCAmPeg3C2-#54 General Method J 12.4 (30%)procedure O MalPeg3C2-#47 General Method J* 19 (62%) procedure DAmPeg6C2-#47 General Method J 50 (77%) procedure NmcValCitPABCAmPeg3C2-#47 General Method J 6.4 (18%) procedure OmcValCitPABCAmPeg6C2-#47 General Method J 18 (50%) procedure OMalPeg3C2-#42 General Method J 22 (70%) procedure D AmPeg6C2-#42 GeneralMethod J 53 (75%) procedure N mcValCitPABCAmPeg6C2-#42 General Method J15.4 (43%) procedure O mcValCitPABCAmPeg3C2-#42 General Method J* 12(26%) procedure O MalPeg3C2-#26 General Method J* 13.8 (51%) procedure Dmc-#41 General Method J* 9.6 (38%) procedure D AmPeg6C2-#26 GeneralMethod J 59 (87%) procedure N mcValCitPABCAmPeg3C2-#26 General Method J23.4 (45%) procedure O MalPeg3C2Va1CitPABC-#26 General Method J 16 (42%)procedure P mcValCitPABCAmPeg6C2-#26 General Method J 15 (38%) procedureO mc-#36 General Method J* 26 (80%) procedure D MalPeg6C2-#54 GeneralMethod J 27 (67%) procedure D MalPeg3C2Va1CitPABC-#47 General Method J17 (49%) procedure P MalPeg3C2-#36 General Method J* 9.2 (33%) procedureD MalPeg6C2-#47 General Method J* 24 (78%) procedure D MalPeg6C2-#26General Method J* 29 (75%) procedure D MalPeg6C2-#36 General Method J 18(58%) procedure D mcValCitPABCAmPeg3C2-#36 General Method J 16 (51%)procedure O AmPeg6C2-#36 General Method J 51 (78%) procedure NmcValCitPABC-#60 General Method J 1.9 (4.3%) procedure EmcValCitPABCAmPeg6C2-#36 General Method J 11.6 (35%) procedure OmcValCitPABCAmPeg3C2-#41 General Method J 5.7 (26%) procedure OMalPeg6C2-#60 General Method J 24 (75%) procedure D AmPeg6C2-#60 GeneralMethod J 31 (80%) procedure N MalPeg3C2-#60 General Method J 17.3 (58%)procedure D MalPeg6C2-#41 General Method J with AcOH as 11 (28%)procedure D modifier AmPeg6C2-#66 General Method J 5 (10%) procedure NmcValCitPABCAmPeg6C2-#60 General Method J 12 (34%) procedure O mc-#70General Method J* 11.2 (46%) procedure D 2AcAmPeg6C2-#66 General MethodJ 8 (60%) procedure Q mc-#66 General Method J with AcOH as 8.2 (32%)procedure D modifier mcValCitPABC-#88 General Method J 7.9 (45%)procedure E mcValCitPABC-#88 General Method J 7.5 (39%) procedure Emc-#92 General Method F 15 (66%) procedure D mcValCitPABC-#44 GeneralMethod F 1.6 (4%) procedure E mc-#108 General Method H without 8.8 (37%)procedure D modifier mcValCitPABC-#108 General Method F 6.1 (14%)procedure E NHSCOPeg2C2ValCitPABC-#66 General Method F* 6.8 (32%)procedure X2 mcValCitPABC-#98 General Method J* 4.8 (11%) procedure EmcValCitPABC-#95 General Method J* 13 (28%) procedure E MalPeg3C2-#69General Method K 12.8 (35%) procedure D AmPeg6C2-#69 General Method J*71 (69%) procedure N mcValCitPABC-#84 General Method J* 4.9 (11%)procedure E AmCapValCitPABC-#54 General Method K* 97 (53%) procedure RmcValCitPABC-#226 General Method J* 7.1 (16%) procedure EmcValCitPABC-#117 General Method J* 15.8 (36%) procedure E MalPeg6C2-#98General Method I* 7.4 (22%) procedure D mcValCitPABC-#118 General MethodJ* 11.7 (29%) procedure E mcValCitPABC-#80 General Method J* 3.8 (12%)procedure E MalPeg6C2-#118 General Method J* 11 (4.5%) procedure DMalPeg6C2-#230 General Method H 2.8 (8%) procedure D mcValCitPABC-#232General Method J* 13.6 (29%) procedure E mc-#117 General Method J* 9.5(40%) procedure D MalPeg6C2-#117 General Method K* 2.3 (8%) procedure Dmv-#69 General Method J* 22 (49%) procedure D mb-#69 General Method J*12 (28%) procedure D AmPeg6C2-#234 General Method J* 16 (52%) procedureN AmPeg6C2-#235 General Method J* 16.5 (70%) procedure N mc-#118 GeneralMethod J* 41 (38%) procedure D MalPeg6C2-#123 General Method J* 85 (40%)procedure D mc-#226 General silica chromatography 290 (40%) procedure Dme-#118 General Method J* 16.2 (42%) procedure D mc-#131 General MethodJ* 16.3 (51%) procedure D mb-#118 General Method J* 7.9 (23%) procedureD mcValCitPABC-#134 General Method J* 17 (33%) procedure E mc-#145General Method K 6 (20%) procedure D MalPeg6C2-#126 General Method J*16.4 (26%) procedure D mc-#126 General Method K* 16.3 (32%) procedure Dmv-#118 General Method J* 11.7 (34%) procedure D mc-#172 General MethodJ* 10 (56%) procedure D MalPeg6C2-#226 General Method K* 15 (10%)procedure D MalPeg6C2-#145 General Method K 1.8 (3.7%) procedure Dmc-#162 General Method H* without 1.2 (3.1%) procedure D modifiermc-#163 General Method K* 9.9 (26%) procedure D mcValCitPABC-#231General Method J* 0.2 (4%) procedure E MalPeg6C2-#238 General silicachromatography 240 (77%) procedure D MalPeg6C2-#239 General mediumpressure C18 104 (39%) procedure D chromatography mc-#123 General silicachromatography 345 (quant.) procedure D MalC6-#54 General Method J* 16.3(30%) procedure S mc-#231 General Method J* 10 (60%) procedure DMalC6-#118 General Method J* 5.3 (10%) procedure S mcValCitPABC-#123General silica chromatography 179 (60%) procedure E mc-#237 GeneralMethod J* 12.6 (47%) procedure D mc-#158 General Method J* 7.1 (28%)procedure D MalC6Am-#151 General Method J* 18.4 (86%) procedure DPFPCOPeg2C2 ValCitPABC-#54 General Method J* AcOH as 70 (68%) procedureX3 modifier mcValCitPABC-#154 General medium pressure C18 10 (19%)procedure E chromatography MalC6Am-#153 General Method K* 18.7 (47%)procedure D PFPCOPeg2C2AmPeg2C2-#69 General Method R* 40 (64%) procedureX4 mcValCitPABC-#246 General medium pressure C18 21 (45%) procedure Echromatography PFPCOPeg2C2AlaAlaAsnPABC-#54 General Method R* 16.8 (54%)procedure X1 PFPCOPeg2C2-#54 General Method R* 4.1 (56%) procedure VPFPCOPeg2C2AmPeg2C2PABC-#54 General Method R* 1.1 (39%) procedure WAmPeg6C2-#115 General Method J* 100 (48%) procedure N PFPCOPeg5C2-#115General Method J* 29 (26%) procedure V mcGly-#201 — silicachromatography 25 (16.2%) AzCOC2Ph4AmCOPeg2C2-#54 General Method R* 3.5(52%) procedure T AzCOC2Ph4AmPeg1C1ValCitPABC- General Method R* 93(43%) #54 procedure U AzCOC2Ph4AmPeg1C1ValCitPABC- General Method R* 1.7(42%) #30 procedure U AzCOC2Ph4AmCOPeg2C2-#69 General Method R* 88 (62%)procedure T AzCOC2Ph4AmCOPeg2C2-#115 General Method R* 75 (82%)procedure T AcLysValCitPABC-#54 General Method J* 86 (40%) procedure X5

TABLE 18B Selected compounds (cytotoxic peptides with linkers) of theinvention Mass spectrum: LC-MS or HPLC observed m/z and retention timein linker with payload # minutes: ESI-MS IUPAC Name mcValCitPABC-#34HPLC (Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M):L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1380.6 [M + Na⁺],(12.899 {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2- minutes)phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl-L- ornithinamideMalPeg3C2-#41 LC-MS: N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-1031.7 [M + H⁺], yl)ethoxy]ethoxy}ethoxy)propanoyl]-N-methyl-L-valyl-1054.8 [M + Na⁺] (0.88N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy- minutes); HPLC2-phenylethyl]amino}-1-methoxy-2-methyl-3- (Protocol D):thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1- 10.559 minutesoxoheptan-4-yl]-N-methyl-L-valinamide MalPeg6C2-#42 LC-MS:N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- 1178.2 [M + H⁺],3,6,9,12,15,18-hexaoxahenicosan-21-yl]-N-methyl-L- 1197.4 [M + Na⁺](3.50 valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes); HPLCmethoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2- (Protocol Q):yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5- 25.235 minutesmethyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-#44 LC-MS:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- 913.7 [M + H⁺](0.85 2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes);{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- HRMS: Calc:methyl-3-thioxopropyl]pyrrolidiN-1-yl}-3-methoxy-5- 913.5103 [M + H⁺],methyl-1-oxoheptaN-4-yl]-N-methyl-L-valinamide Obsd: 913.5103.MalPeg3C2-#44 LC-MS: N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-1003.8 [M + H⁺](0.82yl)ethoxy]ethoxy}ethoxy)propanoyl]-2-methylalanyl-N- minutes); HPLC[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- (Protocol A):phenylethyl]amino}-1-methoxy-2-methyl-3- 1003.5 [M + H⁺],thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1- 1026.4 [M + Na⁺]oxoheptan-4-yl]-N-methyl-L-valinamide (9.095 minutes) MalPeg6C2-#44LC-MS: N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- 1135.8 [M +H⁺] (0.83 3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2- minutes)methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalValCitPABC-#44 LC-MS:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- 1318.9 [M + H⁺](0.89 L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butaN-2-yl]-12- minutes); HPLC(2-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- (Protocol A):phenylethyl]amino}-1-methoxy-2-methyl-3- 1319.6 [M + H⁺],thioxopropyl]pyrrolidiN-1-yl}-2-oxoethyl)-5,5,10- 1342.6 [M + Na⁺]trimethyl-3,6,9-trioxo-8-(propaN-2-yl)-2,13-dioxa- (9.132 minutes)4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl-L- ornithinamide mc-#45LC-MS: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- 927.7 [M +H⁺] (0.92 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes);[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- HRMS: Calc:phenylpropaN-2-yl]amino}-2-methyl-3- 927.5260 [M + H⁺],thioxopropyl]pyrrolidiN-1-yl}-5-methyl-1-oxoheptaN-4- Obsd: 927.5259.yl]-N-methyl-L-valinamide MalPeg3C2-#45 LC-MS:N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- 1017.8 [M + H⁺] (0.90yl)ethoxy]ethoxy}ethoxy)propanoyl]-2-methylalanyl-N- minutes);[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg6C2-#45 LC-MS:N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- 1149.9 [M + H⁺](0.90 3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2- minutes); HPLCmethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- (Protocol A at 45° C.):[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- 1150.5 [M + H⁺],phenylpropan-2-yl]amino}-2-methyl-3- 1171.5 [M + Na⁺]thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4- (9.788 minutes)yl]-N-methyl-L-valinamide mcValCitPABC-#45 LC-MS:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- 1332.8 [M + H⁺](1.86 L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- minutes);HPLC {(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1- (Protocol A at 45°C.): oxo-3-phenylpropan-2-yl]amino}-2-methyl-3- 1333.6 [M + H⁺]thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10- (9.737trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa- minutes)4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl-L- ornithinamidemcValCitPABC-#54 HPLC (Protocol AN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- at 45° C.):L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1342.6 [M + H⁺](9.114 {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)- minutes).2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl- L-ornithinamidemc-#69 LC-MS: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-897.7 [M + H⁺], 2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 919.7(0.81 minutes); {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- HPLC(Protocol A methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5- at 45° C.):methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide 897.5 [M + H⁺] (9.058minutes) MalPeg6C2-#69 HPLC (Protocol AN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- at 45° C.):3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2- 1120.6 [M + H⁺],methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 1142.5 (9.076 minutes){[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABC-#69 HPLC(ProtocolM): N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- 1326.6[M + Na⁺] yl)ethoxy]ethoxy}ethoxy)propanoyl]-N-methyl-L-valyl- (11.962minutes) N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidiN-1-yl}-3-methoxy-5-methyl-1-oxoheptaN-4-yl]-N-methyl-L-valinamide mcValCitPABC-#70 HPLC (Protocol AN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- at 45° C.):L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butaN-2-yl]-12- 1317.6 [M + H⁺](9.282 (2-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1- minutes)oxo-3-phenylpropaN-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidiN-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propaN-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl-L- ornithinamidemcValCitPABC-#75 LC-MS:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- 1273.9 [M + H⁺](0.82 L-valyl-N⁵-carbamoyl-N-{4-[({[3-({(2S)-1- minutes); HPLC[{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1- (Protocol A at 45° C.):methoxy-2-methyl-3-oxo-3-[(2- 1273.6 [M + H⁺], (8.814phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1- minutes)oxoheptan-4-yl}(methyl)amino]-3-methyl-1-oxobutan-2-yl}carbamoyl)oxetan-3- yl]carbamoyl}oxy)methyl]phenyl}-L-ornithinamidemc-#79 HPLC (Protocol AN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- at 45° C.):N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)- 941.5 [M + H⁺],2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- 963.4 [M + Na⁺] (10.444phenylpropaN-2-yl]amino}-2-methyl-3- minutes)thioxopropyl]pyrrolidiN-1-yl}-5-methyl-1-oxoheptaN-4-yl]-N-methyl-L-valinamide mcValCitPABC-#79 HPLC (Protocol A):N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- 1346.6 [M + H⁺],L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- (9.807 minutes){(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,5,5,10-tetramethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl-L- ornithinamidemcValCitPABC-#92 LC-MS:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- 1282.6 [M + H⁺](0.79 L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- minutes);HPLC {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3- (Protocol A at 45°C.): (quinolin-6-ylamino)propyl]pyrrolidin-1-yl}-2- 1282.6 [M + H⁺](7.953 oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)- minutes)2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl-L-ornithinamide mcValCitPABC-#112 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M):L-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1288.6 [M + H⁺],{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- 1310.6 [M + Na⁺] (11.757phenylethyl]amino}-1-methoxy-2-methyl-3- minutes)oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N⁵-carbamoyl-L- ornithinamide mv-#115HPLC (Protocol A N-[5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoyl]-at 45° C.): m/z N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-897.5 [M + H⁺], {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-(9.149 minutes) methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L- valinamide mc-#115 HPLC(Protocol A N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- at 45°C.;) m/z N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3- 911.5 [M +H⁺], {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- (9.676 minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L- valinamide mb-#115 —N-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L- valinamide me-#115 —N-[7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)heptanoyl]-N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L- valinamidemcValCitPABC-#115 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1317.7 [M + H⁺](12.261 {(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,5,5,10-tetramethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mc-#51 HPLC (ProtocolN~2~-[(1-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- M): m/zyl)hexanoyl]amino}cyclopropyl)carbonyl]-N- 934.5 [M + H⁺] (11.94[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy- minutes)2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin- 1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-#47 HPLC (ProtocolN~2~-[(1-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- M): m/zyl)hexanoyl]amino}cyclopentyl)carbonyl]-N- 962.5 [M + H⁺] (13.014[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy- minutes)2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin- 1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-#54 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/z2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 936.5 [M + H⁺] (9.22[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl- minutes)1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mcValCitPABC-#47HPLC (Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M):m/z L-valyl-N~5~-carbamoyl-N-[4-({[(1-{[(2S)-1- 1368.6 [M + H⁺] (13.157{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin- 1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan- 2-yl]carbamoyl}cyclopentyl)carbamoyl]oxy}methyl)phenyl]- L-ornithinamidemcValCitPABC-#26 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/zL-valyl-N-{4-[(5S,8S,11S,12R)-11-[(2S)-butan-2-yl]- 1386.6 [M + H⁺](16.21 12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)- minutes)2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-5,8-di(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemc-#26 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- A*): m/zN-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 980.5 [M + H⁺](10.628 [(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1- minutes)(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mcValCitPABC-#42HPLC (Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-A*): m/z L-valyl-N-{4-[(5S,8S,11S,12R)-11-[(2S)-butan-2-yl]- 1361.7 [M +H⁺] (9.831 12-(2-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1- minutes)methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl- 3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-5,8-di(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemcValCitPABC-#36 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- A*): m/zL-valyl-N-{4-[(5S,8S,11S,12R)-11-[(2S)-butan-2-yl]- 1324.6 [M + Na⁺²³](9.987 12-{2-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2- minutes)phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-2-oxoethyl}-4,10-dimethyl-3,6,9-trioxo-5,8-di(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mc-#42 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- A*): m/zN-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 955.5 [M + H⁺](10.679 [(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- minutes)phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl- L-valinamide AmPeg6C2-#54LC-MS (Protocol N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- H):m/z 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 1078.7 [M + H⁺](2.56 [(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl- minutes)1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide MalPeg3C2-#54LC-MS (Protocol N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- H):m/z yl)ethoxy]ethoxy}ethoxy)propanoyl]-2-methylalanyl-N- 1026.6 [M + H⁺](3.54 [(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy- minutes)2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABCAmPeg6C2- LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #54 H):m/z L-valyl-N-{4-[(30S,33S,34R)-33-[(2S)-butan-2-yl]-34- 1677.9 [M + H⁺](3.48 (2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3- minutes){[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-27,27,32-trimethyl-3,25,28,31-tetraoxo-30-(propan-2-yl)-2,7,10,13,16,19,22,35-octaoxa-4,26,29,32-tetraazahexatriacont-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemcValCitPABCAmPeg3C2- LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #54 H): m/zL-valyl-N-{4-[(21S,24S,25R)-24-[(2S)-butan-2-yl]-25- 1545.8 [M + H⁺](3.48 (2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3- minutes){[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-18,18,23-trimethyl-3,16,19,22-tetraoxo-21-(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide MalPeg3C2-#47 LC-MS (ProtocolN~2~-[(1-{[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H- Q1): m/z pyrrol-1-1052.7 [M + H⁺] (0.88yl)ethoxy]ethoxy}ethoxy)propanoyl]amino}cyclopentyl)carbonyl]- minutes)N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide AmPeg6C2-#47LC-MS (Protocol 1-amino-N-(1-{[(2S)-1-{[(3R,4S,5S)-3-methoxy-1- H): m/z{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)- 1104.88 [M + H⁺](2.65 2-phenyl-1-(1,3-thiazol-2- minutes)yl)ethyl]amino}propyl]prrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}cyclopentyl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide mcValCitPABCAmPeg3C2- LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #47 H):m/z L-valyl-N~5~-carbamoyl-N-(4-{16-[(1-{[(2S)-1- 1571.8 [M + H⁺] (3.56{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3- thiazol-2-yl)ethyl]amino}propyl]prrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}cyclopentyl)amino]-3,16-dioxo-2,7,10,13-tetraoxa-4-azahexadec-1-yl}phenyl)-L- ornithinamidemcValCitPABCAmPeg6C2- HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #47 H): m/zL-valyl-N~5~-carbamoyl-N-(4-{25-[(1-{[(2S)-1- 1703.8 [M + H⁺] (3.57{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3- thiazol-2-yl)ethyl]amino}propyl]prrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}cyclopentyl)amino]-3,25-dioxo-2,7,10,13,16,19,22-heptaoxa-4-azapentacos-1- yl}phenyl)- L-ornithinamideMalPeg3C2-#42 LC-MS (ProtocolN-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- H): m/zyl)ethoxy]ethoxy}ethoxy)propanoyl]-N-methyl-L-valyl- 1045.7 [M + H⁺](3.92 N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2- yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AmPeg6C2-#42 LC-MS(Protocol N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- H): m/zN-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 1097.7 [M + H⁺](2.80 [(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- minutes)phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamidemcValCitPABCAmPeg6C2- LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #42 H): m/zL-valyl-N-{4-[(27S,30S,33S,34R)-33-[(2S)-butan-2-yl]- 1696.8 [M + H⁺](3.73 34-(2-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1- minutes)methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-26,32-dimethyl-3,25,28,31-tetraoxo-27,30-di(propan-2-yl)-2,7,10,13,16,19,22,35-octaoxa-4,26,29,32-tetraazahexatriacont-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemcValCitPABCAmPeg3C2- LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #42 H): m/zL-valyl-N-{4-[(18S,21S,24S,25R)-24-[(2S)-butan-2-yl]- 1564.8 [M + H⁺](3.70 25-(2-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1- minutes)methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-17,23-dimethyl-3,16,19,22-tetraoxo-18,21-di(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide MalPeg3C2-#26 LC-MS (ProtocolN-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- H): m/zyl)ethoxy]ethoxy}ethoxy)propanoyl]-N-methyl-L-valyl- 1070.6 [M + H⁺](3.94 N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-PLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanoyl]-A):N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- m/z{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- 941.5 [M + H⁺] (9.883methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy- minutes)5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide AmPeg6C2-#26 LC-MS(Protocol N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- H): m/zN-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 1122.6 [M + H⁺](2.76 [(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1- minutes)(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamidemcValCitPABCAmPeg3C2- LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #26 H): m/zL-valyl-N-{4-[(18S,21S,24S,25R)-24-[(2S)-butan-2-yl]- 1588.0 [M + H⁺](3.74 25-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)- minutes)2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-17,23-dimethyl-3,16,19,22-tetraoxo-18,21-di(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide MalPeg3C2ValCitPABC-#26 LC-MS(Protocol N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- H): m/zyl)ethoxy]ethoxy}ethoxy)propanoyl]-L-valyl-N-{4- 1476.8 [M + H⁺] (3.81[(5S,8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2- minutes)[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,10-dimethyl-3,6,9-trioxo-5,8-di(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mcValCitPABCAmPeg6C2- LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #26 H):m/z L-valyl-N-{4-[(27S,30S,33S,34R)-33-[(2S)-butan-2-yl]- 1721.9 [M +H⁺] (3.75 34-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)- minutes)2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-26,32-dimethyl-3,25,28,31-tetraoxo-27,30-di(propan-2-yl)-2,7,10,13,16,19,22,35-octaoxa-4,26,29,32-tetraazahexatriacont-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamide mc-#36LC-MS (Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-Q1): m/z N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2- 897.7 [M +H⁺] (1.00 {(1R,2R)-1-methoxy-2-methyl-3-[(2- minutes)phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-5- methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide MalPeg6C2-#54 LC-MS (ProtocolN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- H): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2- 1158.7 [M + H⁺] (3.55methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes)[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl- 1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg3C2ValCitPABC-#47LC-MS (Protocol N-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- H):m/z yl)ethoxy]ethoxy}ethoxy)propanoyl]-L-valyl-N~5~- 1458.7 [M + H⁺](3.56 carbamoyl-N-[4-({[(1-{[(2S)-1-{[(3R,4S,5S)-3- minutes)methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3- oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan- 2-yl]carbamoyl}cyclopentyl)carbamoyl]oxy}methyl)phenyl]- L-ornithinamideMalPeg3C2-#36 LC-MS (ProtocolN-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- H): m/zyl)ethoxy]ethoxy}ethoxy)propanoyl]-N-methyl-L-valyl- 987.7 [M + H⁺](3.97 N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1- minutes)methoxy-2-methyl-3-[(2-phenylethyl)amino]-3- thioxopropyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}-N- methyl-L-valinamide MalPeg6C2-#47LC-MS (Protocol 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(1-{[(2S)-H): m/z 1-{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- 1184.7 [M + H⁺](3.67 methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3- minutes)thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}cyclopentyl)-3,6,9,12,15,18-hexaoxahenicosan-21-amide MalPeg6C2-#26 LC-MS (ProtocolN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- H): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-N-methyl-L- 1202.7 [M + H⁺] (3.93valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg6C2-#36 LC-MS(Protocol N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- H): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-N-methyl-L- 1118.8 [M − H] (3.96valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1- minutes)methoxy-2-methyl-3-[(2-phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl}- N-methyl-L-valinamidemcValCitPABCAmPeg3C2- LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #36 H): m/zL-valyl-N-{4-[(18S,21S,24S,25R)-24-[(2S)-butan-2-yl]- 1506.8 [M + H⁺](3.76 25-{2-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2- minutes)phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-2-oxoethyl}-17,23-dimethyl-3,16,19,22-tetraoxo-18,21-di(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N~5~- carbamoyl-L-ornithinamideAmPeg6C2-#36 LC-MS (ProtocolN-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- H): m/zN-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2- 1039.7 [M + H⁺](2.68 {(1R,2R)-1-methoxy-2-methyl-3-[(2- minutes)phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-5-methyl-1-oxoheptan- 4-yl}-N-methyl-L-valinamide mcValCitPABC-#60 HPLC(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1307.6 [M + H⁺](12.696 {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1- minutes)phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mcValCitPABCAmPeg6C2- LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #36 H):m/z L-valyl-N-{4-[(27S,30S,33S,34R)-33-[(2S)-butan-2-yl]- 1638.0 [M +H⁺] (3.77 34-{2-[(2S)-2-{(1R,2R)-1-methoxy-2-methyl-3-[(2- minutes)phenylethyl)amino]-3-thioxopropyl}pyrrolidin-1-yl]-2-oxoethyl}-26,32-dimethyl-3,25,28,31-tetraoxo-27,30-di(propan-2-yl)-2,7,10,13,16,19,22,35-octaoxa-4,26,29,32-tetraazahexatriacont-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mcValCitPABCAmPeg3C2- LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- #41 H): m/zL-valyl-N-{4-[(18S,21S,24S,25R)-24-[(2S)-butan-2-yl]- 1550.9 [M + H⁺](3.53 25-(2-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-17,23-dimethyl-3,16,19,22-tetraoxo-18,21-di(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide MalPeg6C2-#60 LC-MS (ProtocolN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- H): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2- 1101.8 [M + H⁺] (3.66methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes)[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1-phenylcyclopropyl)methyl]amino}propyl]pyrrolidin- 1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AmPeg6C2-#60 LC-MS (ProtocolN-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- H): m/z2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 1021.7 [M + H⁺] (2.57[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1- minutes)phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg3C2-#60 LC-MS (ProtocolN-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- H): m/zyl)ethoxy]ethoxy}ethoxy)propanoyl]-2-methylalanyl-N- 969.7 [M + H⁺](3.65 [(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy- minutes)2-methyl-3-oxo-3-{[(1- phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4- yl]-N-methyl-L-valinamide MalPeg6C2-#41LC-MS (Protocol N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- H):m/z 3,6,9,12,15,18-hexaoxahenicosan-21-yl]-N-methyl-L- 1163.0 [M − H](3.70 valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1- minutes)carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-thioxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AmPeg6C2-#66 LC-MS (ProtocolN-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- Q1): m/z2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 1009.8 [M + H⁺] (0.72{[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1- minutes)methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mcValCitPABCAmPeg6C2-LC-MS (Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-#60 H): m/z L-valyl-N-{4-[(30S,33S,34R)-33-[(2S)-butan-2-yl]-34- 1621.0[M + H⁺] (3.55 (2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1-minutes) phenylcyclopropyl)methyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-27,27,32-trimethyl-3,25,28,31-tetraoxo-30-(propan-2-yl)-2,7,10,13,16,19,22,35-octaoxa-4,26,29,32-tetraazahexatriacont-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mc-#70 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/z2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 911.5 [M + H⁺] (11.847[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- minutes)phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide 2AcAmPeg6C2-#66LC-MS (Protocol N-(24-bromo-23-oxo-4,7,10,13,16,19-hexaoxa-22- Q1): m/zazatetracosan-1-oyl)-2-methylalanyl-N-[(3R,4S,5S)-1- 1129.8 [M + H⁺](0.85 {(2S)-2-[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1- minutes)yl)ethyl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide mc-#66LC-MS (Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-Q1): m/z 2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 867.7 [M +H⁺] (0.90 {[2-(cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1- minutes)methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mcValCitPABC-#88 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1355.9 [M + H⁺](0.87 {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)- minutes)2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-4,5,5,10-tetramethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemcValCitPABC-#88 LC-MS (ProtocolN-{6-[(bromoacetyl)amino]hexanoyl}-L-valyl-N-{4- Q1): m/z[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2- 1314.9 [M + H⁺] (0.91[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1- minutes)yl)ethyl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mc-#92 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1): m/z2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 876.7 [M + H⁺] (0.75[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-(quinolin-6- minutes)ylamino)propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABC-#44 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- A): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1318.6 [M + H⁺](9.174 {(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3- thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide mc-#108 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- A): m/z2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 909.5 [M + H⁺] (9.063{[(R)-(7S)-bicyclo[4.2.0]octa-1,3,5-trien-7- minutes)yl(carboxy)methyl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4- yl]-N-methyl-L-valinamidemcValCitPABC-#108 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/zL-valyl-N-{4-[(8S,11S,12R)-12-(2-{(2S)-2-[(1R,2R)-3- 1315.7 [M + H⁺](11.89 {[(R)-(7S)-bicyclo[4.2.0]octa-1,3,5-trien-7- minutes)yl(carboxy)methyl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-11-[(2S)-butan-2-yl]-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~- carbamoyl-L-ornithinamideNHSCOPeg2C2ValCitPABC- HPLC (ProtocolN-[3-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3- #66 M): m/zoxopropoxy}ethoxy)propanoyl]-L-valyl-N-{4- 683.3 [M + H⁺²] (10.03[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2- minutes)[(1R,2R)-3-{[2-(cyclohepta-2,4,6-trien-1- yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~- carbamoyl-L-ornithinamidemcValCitPABC-#98 HPLC (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/zL-valyl-N~5~-carbamoyl-N-{4-[({[(2S)-2-{[(2S)-1- 1368.6 [M + H⁺] (12.504{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3- thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpyrrolidin-1-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide mcValCitPABC-#95 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N-{4-[(9S,12S,13R)-12-[(2S)-butan-2-yl]-13-(2- 1356.5 [M + H⁺](1.79 {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)- minutes)2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-6,6,11-trimethyl-3,7,10-trioxo-9-(propan-2-yl)-2,14-dioxa-4,8,11-triazapentadec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamideMalPeg3C2-#69 HPLC (ProtocolN-[3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- M): m/zyl)ethoxy]ethoxy}ethoxy)propanoyl]-2-methylalanyl-N- 987.5 [M + H⁺](10.702 [(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4- yl]-N-methyl-L-valinamideAmPeg6C2-#69 LC-MS (ProtocolN-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- H): m/z2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 1040.1 [M + H⁺] (2.12{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABC-#84 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1371.4 [M + H⁺](1.89 {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S)-2- minutes)phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}-3-thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,5,5,10-tetramethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamideAmCapValCitPABC-#54 LC-MS (ProtocolN-(6-aminohexanoyl)-L-valyl-N-{4-[(8S,11S,12R)-11- H): m/z[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2- 1262.3 [M + H⁺](2.35 methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2- minutes)yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~- carbamoyl-L-ornithinamidemcValCitPABC-#226 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1330.9 [M + H⁺](1.77 {(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1- minutes)oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,5,5,10-tetramethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemcValCitPABC-#117 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N~5~-carbamoyl-N-{4-[({[(2S)-2-{[(2S)-1- 1342.6 [M + H⁺] (1.80{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2- yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpyrrolidin-1-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide MalPeg6C2-#98 HPLC(Protocol 1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- M): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2-methyl-L- 1185.6 [M + H⁺](11.985 prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABC-#118 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N~5~-carbamoyl-N-{4-[({[(2S)-2-{[(2S)-1- 1328.6 [M + H⁺] (1.68{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpyrrolidin-1-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide mcValCitPABC-#80 HPLC(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- M): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1353.6 [M + Na⁺](12.751 {(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3- thioxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-4,5,5,10-tetramethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide MalPeg6C2-#118 LC-MS(Protocol 1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- Q): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2-methyl-L- 1145.6 [M + H⁺] (1.66prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1- minutes)carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg6C2-#230 HPLC (ProtocolN-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- M): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2- 1146.6 [M + H⁺] (12.071methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes){[(R)-carboxy(1-phenylcyclopropyl)methyl]amino}-1- methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABC-#232 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N~5~-carbamoyl-N-{4-[({[(2R)-2-{[(2S)-1- 1367.3 [M + H⁺] (1.81{[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3- thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpyrrolidin-1-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide mc-#117 LC-MS (Protocol1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/z2-methyl-L-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 937.4 [M + H⁺](1.91 [(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- minutes)phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide MalPeg6C2-#117HPLC (Protocol 1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- M):m/z 3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2-methyl-L- 1161.6 [M + H⁺](12.115 prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mv-#69 LC-MS (ProtocolN-[5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoyl]- Q): m/z2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 883.3 [M + H⁺] (1.57{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mb-#69 HPLC (ProtocolN-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]- M): m/z2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 869.5 [M + H⁺](10.874 {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AmPeg6C2-#234 LC-MS(Protocol N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- Q1): m/z2-methylalanyl-N-{(3R,4S,5S)-1-[(2S)-2- 1514.3 [M + H⁺] (0.76{(3R,4R,7S,12S)-7-benzyl-14-[3-chloro-4-(propan-2- minutes)yloxy)phenyl]-4-methyl-12-[4-(8-methylimidazo[1,2- a]pyridin-2-yl)benzyl]-5,8,14-trioxo-2,9-dioxa-6,13-diazatetradecan-3-yl}pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide AmPeg6C2-#235 LC-MS(Protocol N-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- Q1): m/z2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 1280.2 [M + H⁺] (0.87{[(2S)-1-{[4-(5-fluoro-1,3-benzothiazol-2-yl)-2- minutes)methylphenyl]amino}-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mc-#118 LC-MS(Protocol 1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/z2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 923.3 [M + H⁺](1.73 {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg6C2-#123 LC-MS(Protocol N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- Q1): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2- 1175.3 [M + H⁺] (0.99methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes){[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2- yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-#226 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zN,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)- 925.7 [M + H⁺] (1.852-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- minutes)phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide me-#118 LC-MS(Protocol 1-[7-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)heptanoyl]- Q): m/z2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 937.7 [M + H⁺](1.80 {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-#131 LC-MS (Protocol1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/z2-methyl-D-prolyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 937.3 [M + H⁺](1.88 [(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- minutes)phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mb-#118 LC-MS(Protocol 1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]- Q): m/z2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 895.3 [M + H⁺](1.63 {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABC-#134 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N~5~-carbamoyl-N-{4-[({[(2S)-2-{[(2S)-1- 1314.3 [M + H⁺] (1.67{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1- minutes)hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2- methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}-2-methylpyrrolidin-1-yl]carbonyl}oxy)methyl]phenyl}-L-ornithinamide mc-#145 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1): m/zN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)- 897.34 [M + H⁺](0.90 3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1- minutes)methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamideMalPeg6C2-#126 HPLC (Protocol methylN-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[1- M): m/z(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-24,24-dimethyl- 1169.6 [M + Na⁺](12.583 21,25-dioxo-3,6,9,12,15,18-hexaoxa-22-azapentacosan- minutes)25-yl]-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalaninate mc-#126 HPLC (Protocol methylN-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-(3- M): m/z{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- 925.5 [M + H⁺] (12.994yl)hexanoyl]amino}-2,2-dimethylpropanoyl)-L- minutes)valyl](methyl)amino}-3-methoxy-5- methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L-phenylalaninate mv-#118 LC-MS (Protocol1-[5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoyl]- Q): m/z2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 909.2 [M + H⁺](1.68 {[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-#172 LC-MS (Protocolmethyl N-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N- Q1): m/z({(3S)-1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- 941.3 [M + H⁺] (0.96yl)hexanoyl]-3-fluoropyrrolidin-3-yl}carbonyl)-L- minutes)valyl](methyl)amino}-3-methoxy-5- methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L- phenylalaninate MalPeg6C2-#226LC-MS (Protocol N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- Q):m/z 3,6,9,12,15,18-hexaoxahenicosan-21-yl]-N,2- 1147.3 [M + H⁺] (1.76dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes)[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3- phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg6C2-#145 LC-MS(Protocol N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- Q1): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-N,2- 1141.3 [M + Na⁺] (0.87dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes){[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1- methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mc-#162 LC-MS (ProtocolN-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-({(2S)-1-[6- Q): m/z(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-2- 937.3 [M + H⁺] (1.50methylpiperidin-2-yl}carbonyl)-L- minutes)valyl](methyl)amino}-3-methoxy-5- methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L-phenylalanine mc-#163 HPLC (ProtocolN-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-({(2R)-1-[6- A): m/z(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-2- 937.5 [M + H⁺]methylpiperidin-2-yl}carbonyl)-L- (7.855 minutes)valyl](methyl)amino}-3-methoxy-5- methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L-phenylalanine mcValCitPABC-#231 LC-MS(Protocol (N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1):m/z L-valyl-N-{4-[(8S,11S,12R)-12-(2-{(2S)-2- 1640.4 [M + Na⁺²³] (0.94[(3R,4R,7S)-7-benzyl-15-{2-[(3,5-dimethyl-1H-pyrrol- minutes)2-yl-kappaN)methylidene]-2H-pyrrol-5-yl-kappaN}-4- methyl-5,8,13-trioxo-2-oxa-6,9,12-triazapentadecan-3-yl]pyrrolidin-1-yl}-2-oxoethyl)-11-[(2S)-butan-2-yl]-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamidato)(difluoro)boron MalPeg6C2-#238 LC-MS(Protocol N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- Q1): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-N,2- 1173.3 [M + H⁺] (0.96dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes)[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S)-1-oxo-3- phenyl-1-(prop-2-en-1-yloxy)propan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide MalPeg6C2-#239 LC-MS (Protocol1-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-21-oxo- Q): m/z3,6,9,12,15,18-hexaoxahenicosan-21-yl]-2-methyl-L- 1201.3 [M + H⁺] (2.02prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1- minutes)tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mc-#123 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1): m/z2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 953.3 [M + H⁺] (1.04{[(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2- minutes)yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl- L-valinamideMalC6-#54 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-2- Q): m/zmethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2- 922.3 [M + H⁺] (1.50[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl- minutes)1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide mc-#231 LC-MS(Protocol {N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- Q1): m/zyl)hexanoyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2- 1213.3 [M + H⁺](0.98 [(3R,4R,7S)-7-benzyl-15-{2-[(3,5-dimethyl-1H-pyrrol- minutes)2-yl-kappaN)methylidene]-2H-pyrrol-5-yl-kappaN}-4- methyl-5,8,13-trioxo-2-oxa-6,9,12-triazapentadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamidato}(difluoro)boron MalC6-#118 LC-MS (Protocol1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-2- Q1): m/zmethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- 909.3 [M + H⁺] (0.76{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide mcValCitPABC-#123 LC-MS(Protocol N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1): m/zL-valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- 1358.3 [M + H⁺](0.97 {(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-1-oxo-3- minutes)phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemc-#237 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1): m/zN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)- 964.4 [M + H⁺](0.96 3-{[(2S)-3-(1H-indol-3-yl)-1-methoxy-1-oxopropan-2- minutes)yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamidemc-#158 HPLC (Protocol methyl N-[(2R,3R)-3-{(2S)-1-[(3R,4S,5S)-4-{[N-M): m/z ({(2S)-1-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- 951.4 [M + H⁺](12.839 yl)hexanoyl]-2-methylpiperidin-2-yl}carbonyl)-L- minutes)valyl](methyl)amino}-3-methoxy-5- methylheptanoyl]pyrrolidin-2-yl}-3-methoxy-2-methylpropanoyl]-L- phenylalaninate MalC6Am-#151 LC-MS(Protocol 1,2-dimethyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2- Q): m/z[(1R,2R)-3-{[(2S)-1-{[6-(2,5-dioxo-2,5-dihydro-1H- 922.3 [M + H⁺] (1.43pyrrol-1-yl)hexyl]amino}-1-oxo-3-phenylpropan-2- minutes)yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin- 1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamidePFPCOPeg2C2ValCitPABC- LC-MS (Protocol N-(3-{2-[3-oxo-3- #54 Q): m/z(pentafluorophenoxy)propoxy]ethoxy}propanoyl)-L- 1502.8 [M + H⁺] (1.98valyl-N-{4-[(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2- minutes){(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamidemcValCitPABC-#154 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q1): m/zL-valyl-N-{4-[(9S,12S,13R)-12-[(2S)-butan-2-yl]-13-(2- 1370.2 [M + H⁺](0.93 {(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)- minutes)2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-4,6,6,11-tetramethyl-3,7,10-trioxo-9-(propan-2-yl)-2,14-dioxa-4,8,11-triazapentadec-1-yl]phenyl}-N~5~-carbamoyl-L- ornithinamide MalC6Am-#153HPLC (Protocol 1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2- A): m/z[(1R,2R)-3-{[(2S)-1-{[6-(2,5-dioxo-2,5-dihydro-1H- 922.5 [M + H⁺] (7.352pyrrol-1-yl)hexyl]amino}-1-oxo-3-phenylpropan-2- minutes)yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin- 1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamidePFPCOPeg2C2AmPeg2C2-#69 HPLC (ProtocolN-[11,20-dioxo-20-(pentafluorophenoxy)-4,7,14,17- BB): m/ztetraoxa-10-azaicosan-1-oyl]-2-methylalanyl-N- 1217.6 [M + H⁺] (12.936[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamidemcValCitPABC-#246 LC-MS (ProtocolN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]- Q): m/zL-valyl-N-(4-{(6S,9R,10R)-6-benzyl-10-[(2S)-1- 1327.9 [M + H⁺] (1.36{(3R,4S,5S)-4-[(1,2-dimethyl-L-prolyl-L- minutes)valyl)(methyl)amino]-3-methoxy-5- methylheptanoyl}pyrrolidin-2-yl]-9-methyl-3,8-dioxo-2,11-dioxa-4,7-diazadodec-1-yl}phenyl)-N~5~-carbamoyl-L-ornithinamide PFPCOPeg2C2AlaAlaAsnPABC- HPLC(Protocol N-(3-{2-[3-oxo-3- #54 AB): m/z(pentafluorophenoxy)propoxy]ethoxy}propanoyl)-L- 1503.6 [M + H⁺] (8.06alanyl-L-alanyl-N~1~-{4-[(8S,11S,12R)-11-[(2S)- minutes)butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-L- aspartamidePFPCOPeg2C2-#54 HPLC (Protocol 2-methyl-N-(3-{2-[3-oxo-3- AB): m/z(pentafluorophenoxy)propoxy]ethoxy}propanoyl)alanyl- 1098.4 [M + H⁺](8.44 N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1- minutes)methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamidePFPCOPeg2C2AmPeg2C2PABC- HPLC (ProtocolN-{[(4-{[11,20-dioxo-20-(pentafluorophenoxy)- #54 AB): m/z4,7,14,17-tetraoxa-10-azaicosan-1- 1428.2 [M + Na⁺] (10.32oyl]amino}benzyl)oxy]carbonyl}-2-methylalanyl-N- minutes)[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)- 2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AmPeg6C2-#115 HPLC (ProtocolN-(21-amino-4,7,10,13,16,19-hexaoxahenicosan-1-oyl)- A): m/zN,2-dimethylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)- 1053.5 [M + H⁺](7.35 3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2- minutes)methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide PFPCOPeg5C2-#115 LC-MS(Protocol N,2-dimethyl-N-[19-oxo-19-(pentafluorophenoxy)- Q): m/z4,7,10,13,16-pentaoxanonadecan-1-oyl]alanyl-N- 1205.1 [M + H⁺] (1.99[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2- minutes)phenylethyl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamidemcGly-#201 HPLC (Protocol 1,2-dimethyl-D-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-EB): (4.0 minutes):[(1R,2R)-3-({(2S)-3-[4-({N-[6-(2,5-dioxo-2,5-dihydro- ESI-MS m/z1H-pyrrol-1-yl)hexanoyl]glycyl}amino)phenyl]-1- 1023.59 [M + H⁺]methoxy-1-oxopropan-2-yl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin- 1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AzCOC2Ph4AmCOPeg2C2- HPLC(Protocol 2-methyl-N-(3-{2-[3-oxo-3-({4-[3-oxo-3-(2- #54 FB): m/zoxoazetidin-1- 1132.4 [M + H⁺] (10.18yl)propyl]phenyl}amino)propoxy]ethoxy}propanoyl)alanyl- minutes)N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AzCOC2Ph4AmPeg1C1ValCitHPLC (Protocol N-{[2-oxo-2-({4-[3-oxo-3-(2-oxoazetidin-1- PABC-#54 FB):m/z yl)propyl]phenyl}amino)ethoxy]acetyl}-L-valyl-N-{4- 1465.8 [M + H⁺](10.97 [(8S,11S,12R)-11-[(2S)-butan-2-yl]-12-(2-{(2S)-2- minutes)[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide AzCOC2Ph4AmPeg1C1ValCit HPLC (ProtocolN-{[2-oxo-2-({4-[3-oxo-3-(2-oxoazetidin-1- PABC-#30 FB): m/zyl)propyl]phenyl}amino)ethoxy]acetyl}-L-valyl-N~5~- 1491.8 [M + H⁺](10.56 carbamoyl-N-[4-({[(1-{[(2S)-1-{[(3R,4S,5S)-3- minutes)methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methyl-1-oxobutan- 2-yl]carbamoyl}cyclopentyl)carbamoyl]oxy}methyl)phenyl]- L-ornithinamideAzCOC2Ph4AmCOPeg2C2- HPLC (Protocol2-methyl-N-(3-{2-[3-oxo-3-({4-[3-oxo-3-(2- #69 AB): m/z oxoazetidin-1-1065.3 [M + H⁺] (12.02yl)propyl]phenyl}amino)propoxy]ethoxy}propanoyl)alanyl- minutes)N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AzCOC2Ph4AmCOPeg2C2- HPLC(Protocol N,2-dimethyl-N-(3-{2-[3-oxo-3-({4-[3-oxo-3-(2- #115 AA): m/zoxoazetidin-1- 1078.6 [M + H⁺] (12.02yl)propyl]phenyl}amino)propoxy]ethoxy}propanoyl)alanyl- minutes)N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide AcLysValCitPABC-#54 LC-MS(Protocol N~2~-acetyl-L-lysyl-L-valyl-N-{4-[(8S,11S,12R)-11- Q): m/z[(2S)-butan-2-yl]-12-(2-{(2S)-2-[(1R,2R)-1-methoxy-2- 1319.3 [M + H⁺²](1.34 methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2- minutes)yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-5,5,10-trimethyl-3,6,9-trioxo-8-(propan-2-yl)-2,13-dioxa-4,7,10-triazatetradec-1-yl]phenyl}-N~5~-carbamoyl-L-ornithinamide, trifluoroacetic acid salt

TABLE 19A Selected conjugates of the invention Theoretical Δ Amount of2,2′,2″- mass or phosphanetriyltripropanoic payload/linker Prep.acid/Linker-Payload or molecular ADC-Linker-Payload # Method TCEP/PL(x/y) weight H-(C)_MalPeg3C2-#41 General 2.3/7.5 1031 procedure FH-(C)_MalPeg6C2-#42 General 2.3/7.5 1177 procedure F H-(C)_mc-#44General 2.3/7.5 913 procedure F H-(C)_MalPeg3C2-#44 General 2.2/7   1003procedure F H-(C)_MalPeg6C2-#44 General 2.0/7   1135 procedure FH-(C)_mcValCitPABC-#44 General 2.5/7.5 1319 procedure FH-(C)_Mal-PEG3C2-#45 General 2.3/7.5 1017 procedure FH-(C)_Mal-PEG6C2-#45 General 2.05/10   1149 procedure FH-(C)_mcValCitPABC-#45 General 2.5/10  1342 procedure F H-(C)_mc-#54General 2.2/7.5 897 procedure F H-(C)_Mal-PEG6C2-#69 General 2.1/7.51119 procedure F H-(C)_mcValCitPABC-#69 General 2.5/7.5 1303 procedure FH-(C)_mcValCitPABC-#70 General 2.0/7   1317 procedure F H-(C)_mc-#79General 2.0/7   941 procedure F H-(C)_mcValCitPABC-#79 General 2.3/7.51345 procedure F H-(C)_mc-#115 General 2.2/6.5 911 procedure FH-A114C-(C114)_mc-#51 General NA 934.21 procedure GH-A114C-(C114)_mc-#47 General NA 962.27 procedure GH-A114C-(C114)_mc-#54 General NA 936.2 procedure GH-A114C-(C114)_mcValCitPABC- General 50/10 1367.72 #47 procedure HH-A114C-(C114)_mcValCitPABC- General 100/10  1341.68 #54 procedure HH-A114C-(C114)_mcValCitPABC- General 50/10 1385.8 #26 procedure HH-A114C-(C114)_mc-#26 General 50/10 980.35 procedure HH-A114C-(C114)_mcValCitPABC- General 50/10 1302.69 #36 procedure HH-A114C-(C114)_mcValCitPABC- General 100/7  1360.73 #42 procedure HH-A114C-(C114)_mc-#42 General 50/10 955.27 procedure HH-(C)-mcValCitPABC-#54 General 2.5/10  1342 procedure FH-(C)_mcValCitPABCAmPeg3C2- General 2.5/9   1544 #54 procedure FH-(C)_mcValCitPABCAmPeg6C2- General 2.6/10  1677 #54 procedure FH-(C)_mc-#47 General 1.9/10  962.27 procedure F H-(C)_MalPeg3C2-#54General 1.9/10  1026 procedure F H-(C)_mc-#54 General 2.07/10   936.2procedure F H-(C)_mcValCitPABCAmPeg3C2- General 2.3/7.5 1569.96 #47procedure F H-(C)_MalPeg3C2-#47 General 2.3/7.5 1052 procedure FH-(C)_mcValCitPABCAmPeg3C2- General 2.5/7.5 1562.87 #42 procedure FH-(C)_mc-#41 General 2.5/10  941.24 procedure FH-(C)_mcValCitPABCAmPeg3C2- General 2.5/10  1589.04 #26 procedure FH-(C)_mcValCitPABCAmPeg6C2- General 2.4/7   1701.9 #47 procedure FH-(C)_MalPeg3C2-#42 General 2.3/7   1044.58 procedure FH-(C)_mcValCitPABCAmPeg6C2- General 2.5/7.5 1719.9 #26 procedure FH-(C)_mcValCitPABCAmPeg6C2- General 2.3/7.5 1696.1 #42 procedure FH-(C)_MalPeg6C2-#54 General 2.3/7.5 1158.5 procedure FH-(C)_MalPeg6C2-#47 General 2.3/7.5 1184.5 procedure FH-(C)_MalPeg6C2-#26 General 2.3/7.5 1202.6 procedure FH-(C)-MalPeg6C2-#42 General 2.3/7.5 1177 procedure F H-(C)_mc-#36General 2.3/7.5 896 procedure F H-(C)_mcValCitPABC-#60 General 3.0/7  1284.61 procedure F H-(C)_MalPeg3C2-#26 General 2.5/10  1070.42procedure F H-(C)_mcValCitPABCAmPeg3C2- General 3.0/10  1505.93 #36procedure F H-A114C- General 50/10 1505.93 (C114)_mcValCitPABCAmPeg3C2-procedure H #36 H-A114C-(C114)_MalPeg6C2-#54 General 50/10 1158.5procedure H H-(C)_MalPeg3C2-#60 General 2.3/7.5 969.23 procedure FH-(C)_MalPeg6C2-#60 General 2.3/7.5 1101.4 procedure FH-(C)_MalPeg6C2-#41 General 2.3/7.5 1163.5 procedure F H-(C)-mc-#69General 2.2/7.5 897 procedure F H-(C)_MalPeg3C2-#36 General 2.15/10  987.31 procedure F H-(C)_mcValCitPABCAmPeg6C2- General 2.25/10   1636#36 procedure F H-(C)_MalPeg6C2-#36 General 2.15/10   1119.5 procedure FH-(C)_mcValCitPABCAmPeg3C2- General 2.5/10  1549.94 #41 procedure FH-(C)-MalPeg3C2-#41 General 2.3/7.5 1031 procedure FH-(C)_mcValCitPABCAmPeg6C2- General 2.5/10  1620 #60 procedure FH-A114C-(C114)_mc-#66 General 50/7  866.5 procedure H H-L398C + L443C-General 50/7  1341.68 (C398 + C443)_mcValCitPABC-#54 procedure HH-K392C + L443C- General 100/10  1341 (C392 + C443)_mcValCitPABC-#54procedure H H-L443C-(C443)_mcValCitPABC- General 100/10  1341 #54procedure H H-L398C + V422C- General 50/7  1341.68 (C398 +C422)_mcValCitPABC-#54 procedure H H-(C)-mc-#44 General 2.3/7.5 913procedure F H-(C)-Mal-PEG3C2-#45 General 2.3/7.5 1017 procedure FH-(C)_2AcAmPeg6C2-#66 General 2.4/10  1049.6 procedure FH-(C)-Mal-PEG6C2-#45 General 2.05/10   1149 procedure F H-(C)-mc-#79General 2.0/7   941 procedure F H-(C)-MalPeg3C2-#44 General 2.2/7   1003procedure F H-(C)-mcValCitPABC-#70 General 2.0/7   1317 procedure FH-(C)-MalPeg6C2-#44 General 2.0/7   1135 procedure FH-A114C-(C114)_mcValCitPABC- General  100/8.25 1302 #69 procedure HH-(C)-mcValCitPABC-#79 General 2.3/7.5 1345 procedure FH-A114C-(C114)_mcValCitPABC- General 100/7.5  1346 #79 procedure HH-(C)-mcValCitPABC-#44 General 2.5/7.5 1319 procedure FH-A114C-(C114)_mcValCitPABC- General 100/7.5  1355 #88 procedure HH-(C)-mcValCitPABC-#69 General 2.5/7.5 1303 procedure FH-(C)_2AcAmCapValCitPABC-#66 General 2.2/10  1313.49 procedure FH-A114C-(C114)_mcValCitPABC- General 100/7.5  1331.7 #45 procedure HH-A114C-(C114)_mcValCitPABC- General 100/10  1357 #34 procedure HH-A114C-(C114)_mc-#45 General 100/10  926 procedure HH-A114C-(C114)_mc-#70 General 100/10  911.15 procedure HH-(C)_mcValCitPABC-#112 General 2.3/7.5 1288 procedure FH-(C)-Mal-PEG6C2-#69 General 2.1/7.5 1119 procedure FH-Q347C-(C347)_mcValCitPABC- General 100/10  1302 #69 procedure HH-Y373C-(C373)_mcValCitPABC- General 100/10  1302 #69 procedure HH-E388C-(C388)_mcValCitPABC- General 100/10  1302 #69 procedure HH-N421C-(C421)_mcValCitPABC- General 100/10  1302 #69 procedure HH-L443C-(C443)_mcValCitPABC- General 100/10  1302 #69 procedure HH-L443C-(C443)_mcValCitPABC- General 100/10  1346 #79 procedure HH-A114C-(C114)_mcValCitPABC- General 100/10  1354 #95 procedure HH-A114C-(C114)_mcValCitPABC- General 100/10  1367 #98 procedure HH-A114C-(C114)_MalPeg3C2-#69 General 100/10  987.2 procedure HH-N297Q-(Q)_AmPeg6C2-#42 General NA 1080 procedure KH-N297Q-(Q)_AmPeg6C2-#54 General NA 1061 procedure KH-N297Q-(Q)_AmPeg6C2-#47 General NA 1087 procedure KH-N297Q-(Q)_AmPeg6C2-#36 General NA 1022 procedure KH-N297Q-(Q)_AmPeg6C2-#26 General NA 1105 procedure KH-N297Q-(Q)_AmPeg6C2-#66 General NA 992 procedure KH-L443C-(C443)_MalPeg6C2-#69 General 100/10  1119 procedure HH-Q347C-(C347)_MalPeg6C2-#69 General 100/10  1119 procedure HH-E388C-(C388)_MalPeg6C2-#69 General 100/10  1119 procedure HH-N421C-(C421)_MalPeg6C2-#69 General 100/10  1119 procedure HH-E380C-(C380)_MalPeg6C2-#69 General 100/10  1119 procedure H H-L398C +L443C- General 100/10  1119 (C398 + C443)_MalPeg6C2-#69 procedure HH-K392C + L443C- General 100/10  1119 (C392 + C443)_MalPeg6C2-#69procedure H H-kA111C-(kC111)_MalPeg6C2-#69 General 100/10  1119procedure H H-kK183C-(kC183)_MalPeg6C2-#69 General 100/10  1119procedure H H-kK207C-(kC207)_MalPeg6C2-#69 General 100/10  1119procedure H H-A114C-(C114)_mcValCitPABC- General 100/10  1314.59 #108procedure H H-A114C-(C114)_mcValCitPABC- General 100/10  1371 #84procedure H H-A114C-(C114)_mcValCitPABC- General 100/10  1330 #226procedure H H-A114C-(C114)_mc-#108 General 100/10  909.12 procedure HH-A114C-(C114)_mcValCitPABC- General 100/10  1342 #117 procedure HH-A114C-(C114)_mcValCitPABC- General 100/10  1316 #115 procedure HH-A114C-(C114)_MalPeg6C2-#98 General 100/10  1184 procedure HIL13Ra2-AB08-v1010-hG1- General 2.2/7   1341.68 (C)_mcValCitPABC-#54procedure F IL13Ra2-AB08-v1010-hG1-(C)_mc- General 2.3/7   897.12 #69procedure F IL13Ra2-AB08-v1010-hG1- General 2.3/8   1119(C)_MalPeg6C2-#69 procedure F IL13Ra2-AB08-v1010-hG1- General 2.5/8  1302 (C)_mcValCitPABC-#69 procedure F H-A114C-(C114)_MalPeg6C2-0#118General 100/15  1145 procedure H H-A114C-(C114)_mcValCitPABC- General100/15  1328 0#118 procedure H H-A114C-(C114)_mcValCitPABC- General100/15  1332 #80 procedure H H-A114C-(C114)_mc-#117 General 100/15  937procedure H H-A114C-(C114)_mcValCitPABC- General 100/15  1366 #232procedure H H-A114C-(C114)_MalPeg6C2-#230 General 100/15  1145 procedureH H-A114C-(C114)_MalPeg6C2-#117 General 100/15  1159 procedure HH-A114C-(C114)_mc-#115 General 100/10  911 procedure HH-A114C-(C114)_mv-#115 General 100/10  897 procedure HH-A114C-(C114)_mb-#69 General 100/15  869 procedure HH-A114C-(C114)_mv-#69 General 100/15  883 procedure HH-A114C-(C114)_mc-0#118 General 100/15  923 procedure H H-(C)_mc-#117General 2.0/6.5 937 procedure F H-(C)_MalPeg6C2-#117 General 2.05/6.5 1159 procedure F H-(C)_mc-0#118 General 2.1/7   923 procedure FH-(C)_MalPeg6C2-0#118 General 2.2/7.5 1145 procedure FIL13Ra2-AB08-v1010-hG1-(C)_mc- General 2.35/7   923 0#118 procedure FIL13Ra2-AB08-v1010-hG1-(C)_mc- General 3.0/10  925 #226 procedure FIL13Ra2-AB08-v1010-hG1-(C)_mc- General 3.0/10  937 #117 procedure FIL13Ra2-AB08-v1010-hG1- General 3.0/10  1159 (C)_MalPeg6C2-#117procedure F IL13Ra2-AB08-v1010-hG1- General 3.0/10  1145(C)_MalPeg6C2-0#118 procedure F H-A114C-(C114)_MalPeg6C2-#226 General100/10  1147 procedure H H-A114C-(C114)_mc-#172 General 100/15  940.53procedure H H-A114C-(C114)_mb-0#118 General 100/15  895 procedure HH-A114C-(C114)_me-0#118 General 100/15  937 procedure HH-A114C-(C114)_mcValCitPABC- General 100/15  1314 #134 procedure HH-A114C-(C114)_mc-#131 General 100/15  937 procedure HH-A114C-(C114)_MalPeg6C2-#126 General 100/15  1147 procedure HH-A114C-(C114)_MalPeg6C2-#123 General 100/15  1174 procedure HH-A114C-(C114)_mc-#126 General 100/15  925 procedure HH-A114C-(C114)_mv-0#118 General 100/15  909 procedure HH-(C)_MalPeg6C2-#226 General 2.4/7   1147 procedure F H-(C)_mc-#226General 2.4/7   925 procedure F IL13Ra2-AB08-v1010-hG1- General 3.0/10 1147 (C)_MalPeg6C2-#226 procedure F Notch-28-cG1-(C)_mc-0#118 General2.5/7   923 procedure F Notch-28-cG1-(C)_mc-#115 General 2.35/7   911procedure F Notch-28-cG1-(C)_MalPeg6C2-0#118 General 2.35/7   1145procedure F Notch-28-cG1-(C)_me-0#118 General 2.4/7   937 procedure FNotch-75-cG1-(C)_mc-0#118 General 2.5/7   923 procedure FIL13Ra2-19F9-cG1- General 2.5/8   1341.68 (C)_mcValCitPABC-#54 procedureF IL13Ra2-19F9-cG1- General 2.5/7   1288 (C)_mcValCitPABC-#112 procedureF Notch-28-cG1-(C)_mcValCitPABC- General 2.3/7   1288 #112 procedure FNotch-28-cG1-(C)_MalPeg6C2-#69 General 2.4/7   1119 procedure FNotch-75-cG1-(C)_MalPeg6C2-#69 General 2.4/7   1119 procedure FH-(C)_m(H2O)c-0#118 General 2.35/7   941 procedure IH-(C)_Mal(H2O)Peg6C2-0#118 General 2.35/7   1163 procedure IH-(C)_Mal(H2O)Peg6C2-#69 General 2.1/7   1137 procedure IH-(C)_m(H2O)c-#69 General 2.4/7   915 procedure I H-(C)_me-0#118 General2.2/7   937 procedure F H-(C)_mv-0#118 General 2.2/7   909 procedure FH-(C)_mb-0#118 General 2.1/7   895 procedure F H-A114C-(C114)_MalC6-#54General 100/5  922.22 procedure H H-A114C-(C114)_mc-#231 General 100/5 1213 procedure H H-A114C-(C114)_MalC6-0#118 General 100/5  909.18procedure H H-(C)_Mal(H2O)Peg6C2-#115 General 2.4/7   1151 procedure IH-A114C-(C114)_mc-#158 General 100/10  951 procedure HH-A114C-(C114)_mcValCitPABC- General 100/10  1617 #231 procedure HH-(C)_m(H2O)c-#115 General 2.4/7   929 procedure INotch-75-cG1-(C)_mc-#115 General 3.0/7.0 911.15 procedure FNotch-75-cG1-(C)_me-0#118 General 3.0/7.0 937 procedure FNotch-75-cG1-(C)_MalPeg6C2-0#118 General 3.0/7.0 1144 procedure FH-A114C-(C114)_mc-#237 General 100/10  963 procedure HH-A114C-(C114)_mc-#145 General 100/10  897 procedure HH-A114C-(C114)_MalPeg6C2-#145 General 100/10  1119 procedure HH-A114C-(C114)_mc-#162 General 100/10  937 procedure HH-A114C-(C114)_MalC6Am-#151 General 100/10  905 procedure HNotch-28-cG1-(C)_m(H2O)c-0#118 General 2.2/7   941 procedure INotch-75-cG1-(C)_m(H2O)c-0#118 General 2.2/7   941 procedure IH-(kK188)_COPeg2C2ValCitPABC- General — 1318 #54 procedure JIL13Ra2-AB08-v1010-hG1- General 2.3/7   1163 (C)_Mal(H2O)Peg6C2-0#118procedure F IL13Ra2-AB08-v1010-hG1- General 2.3/7   1151(C)_Mal(H2O)Peg6C2-#115 procedure F IL13Ra2-AB08-v1010-hG1-(C)_mc-General 2.35/7   911 #115 procedure F IL13Ra2-AB08-v1010-hG1- General2.8/7  941 (C)_m(H2O)c-0#118 procedure F H-(C)_mcValCitPABC-0#118General 2.2/7   1328 procedure F IL13Ra2-AB08-v1010-hG1- General2.35/7   929 (C)_m(H2O)c-#115 procedure F H-A114C-(C114)_mcValCitPABC-General 100/5  1369 #154 procedure H H-A114C-(C114)_MalC6Am-#153 General100/10  921 procedure H IL13Ra2-AB08-v1010- General 100/10  1341.68Q347C + kK183C-hG1- procedure H (C347 + kC183)_mcValCitPABC-#54IL13Ra2-AB08-v1010-Q347C-hG1- General 100/10  1341.68(C347)_mcValCitPABC-#54 procedure H IL13Ra2-AB08-v1010-hG1- General —1032 (kK188)_COPeg2C2AmPeg2C2-#69 procedure J IL13Ra2-AB08-v1010-hG1-General — 1318 (kK188)_COPeg2C2ValCitPABC-#54 procedure JIL13Ra2-AB08-v1010-L443C-hG1- General 100/10  1341.68(C443)_mcValCitPABC-#54 procedure H IL13Ra2-AB08-v1010- General 100/10 1341.68 K392C + L443C-hG1- procedure H (C392 + C443)_mcValCitPABC-#54IL13Ra2-AB08-v1010- General 100/10  1341.68 L443C + kK183C-hG1-procedure H (C443 + kC183)_mcValCitPABC-#54 H-(C)_mcValCitPABC-#98General 2.2/7   1367 procedure F H-A114C-(C114)_mcValCitPABC- General100/10  1327 #246 procedure H H-H435A-(C)_mcValCitPABC-#54 General2.2/8   1341.7 procedure F H-M428L + N434S- General 2.2/8   1316(C)_mcValCitPABC-#70 procedure F H-M428L + N434S- General 2.2/8   1341.7(C)_mcValCitPABC-#54 procedure F H-E388C + N421C- General 100/10  1341(C388 + C421)_mcValCitPABC-#54 procedure H H-Q347C + K392C- General100/10  1341 (C347 + C392)_mcValCitPABC-#54 procedure H H-L443C +kK183C- General 100/10  1341 (C443 + kC183)_mcValCitPABC-#54 procedure HH-Q347C + kK183C- General 100/10  1341 (C347 + kC183)_mcValCitPABC-#54procedure H H-Q347C-(C347)_mcValCitPABC- General 100/10  1341 #54procedure H H-K392C + L443C-(C392 + C443)_mc- General 100/10  911 #115procedure H H-E388C + N421C-(C388 + C421)_mc- General 100/10  911 #115procedure H H-Q347C + K392C-(C347 + C392)_mc- General 100/10  911 #115procedure H H-L443C + kK183C- General 100/10  911 (C443 + kC183)_mc-#115procedure H H-Q347C + kK183C- General 100/10  911 (C347 + kC183)_mc-#115procedure H H-Q347C-(C347)_mc-#115 General 100/10  911 procedure HH-kK183C-(kC183)_mcValCitPABC- General 100/10  1341 #54 procedure HH-E388C-(C388)_mcValCitPABC- General 100/10  1341 #54 procedure HH-kK183C-(kC183)_mc-#115 General 100/10  911 procedure HH-E388C-(C388)_mc-#115 General 100/10  911 procedure HH-L443C-(C443)_mc-#115 General 100/10  911 procedure HH-N421C-(C421)_mcValCitPABC- General 100/10  1341 #54 procedure HH-N421C-(C421)_mc-#115 General 100/10  911 procedure HH-A114C-(C114)_mcGly-#201 General 100/10  1023 procedure G

TABLE 19B Selected conjugates of the invention Mass spectra: HPLC-SECretention time and HPLC Δ mass for the Heavy Chain (HC) portion Loadingor Drug (up to 6 Da difference with theoretical Δ per AntibodyADC-Linker-Payload # mass) ratio (DAR) H-(C)_MalPeg3C2-#41 SEC (ProtocolO): 7.317 minutes; HPLC 4.3 (Protocol P): HC Δ mass = 1032H-(C)_MalPeg6C2-#42 SEC (Protocol O): 7.177 minutes; HPLC 3.9 (ProtocolP): HC Δ mass = 1180 H-(C)_mc-#44 SEC (Protocol O): 7.195 minutes; HPLC4.4 (Protocol P): HC Δ mass = 915 H-(C)_MalPeg3C2-#44 SEC (Protocol O):7.247 minutes; HPLC 3.4 (Protocol P): HC Δ mass = 1005H-(C)_MalPeg6C2-#44 SEC (Protocol O): 7.237 minutes; HPLC 3.4 (ProtocolP): HC Δ mass = 1135 H-(C)_mcValCitPABC-#44 SEC (Protocol O): 7.351minutes; HPLC 4.2 (Protocol P): HC Δ mass = 1321 H-(C)_Mal-PEG3C2-#45SEC (Protocol O): 7.364 minutes; HPLC 4.3 (Protocol P): HC Δ mass = 1017H-(C)_Mal-PEG6C2-#45 SEC (Protocol O): 7.419 minutes; HPLC 3.9 (ProtocolP): HC Δ mass = 1154 H-(C)_mcValCitPABC-#45 SEC (Protocol O): 7.159minutes; HPLC 4.1 (Protocol P): HC Δ mass = 1343 H-(C)_mc-#54 SEC(Protocol O): 7.192 minutes; HPLC 4.5 (Protocol P): HC Δ mass = 899H-(C)_Mal-PEG6C2-#69 SEC (Protocol O): 7.350 minutes; HPLC 3.4 (ProtocolP): HC Δ mass = 1122 H-(C)_mcValCitPABC-#69 SEC (Protocol O): 7.254minutes; HPLC 4.5 (Protocol P): HC Δ mass = 1305 H-(C)_mcValCitPABC-#70SEC (Protocol O): 7.466 minutes; HPLC 3.7 (Protocol P): HC Δ mass = 1318H-(C)_mc-#79 SEC (Protocol O): 7.478 minutes; HPLC 4 (Protocol P): HC Δmass = 946 H-(C)_mcValCitPABC-#79 SEC (Protocol O): 7.635 minutes; HPLC3.7 (Protocol P): HC Δ mass = 1349 H-(C)_mc-#115 SEC (Protocol O): 7.510minutes; HPLC 3.54 (Protocol P): HC Δ mass = 912 H-A114C-(C114)_mc-#51 —2.4 H-A114C-(C114)_mc-#47 — 2.3 H-A114C-(C114)_mc-#54 — 2.3H-A114C-(C114)_mcValCitPABC-#47 — 2 H-A114C-(C114)_mcValCitPABC-#54 —1.9 H-A114C-(C114)_mcValCitPABC-#26 — 2 H-A114C-(C114)_mc-#26 — 1.9H-A114C-(C114)_mcValCitPABC-#36 — 2 H-A114C-(C114)_mcValCitPABC-#42 SEC(Protocol P): 7.681 minutes; HPLC 1.95 (Protocol O): HC Δ mass = 1378H-A114C-(C114)_mc-#42 — 2 H-(C)-mcValCitPABC-#54 SEC (Protocol P): 7.159minutes; HPLC 4.1 (Protocol O): HC Δ mass = 1343H-(C)_mcValCitPABCAmPeg3C2-#54 — 4.6 H-(C)_mcValCitPABCAmPeg6C2-#54 —4.5 H-(C)_mc-#47 — 4.2 H-(C)_MalPeg3C2-#54 SEC (Protocol P): 7.179minutes; HPLC 3.7 (Protocol O): HC Δ mass = 1028 H-(C)_mc-#54 — 4H-(C)_mcValCitPABCAmPeg3C2-#47 — 3.7 H-(C)_MalPeg3C2-#47 — 4.3H-(C)_mcValCitPABCAmPeg3C2-#42 — 4.3 H-(C)_mc-#41 — 3.1H-(C)_mcValCitPABCAmPeg3C2-#26 — 3 H-(C)_mcValCitPABCAmPeg6C2-#47 — 4.2H-(C)_MalPeg3C2-#42 SEC (Protocol P): 7.142 minutes; HPLC 4.3 (ProtocolO): HC Δ mass = 1050 H-(C)_mcValCitPABCAmPeg6C2-#26 4.2H-(C)_mcValCitPABCAmPeg6C2-#42 — 4.1 H-(C)_MalPeg6C2-#54 SEC (ProtocolP): 7.254 minutes; HPLC 4.4 (Protocol O): HC Δ mass = 1161H-(C)_MalPeg6C2-#47 SEC (Protocol P): 7.303 minutes; HPLC 4.4 (ProtocolO): HC Δ mass = 1182 H-(C)_MalPeg6C2-#26 — 4.1 H-(C)-MalPeg6C2-#42 SEC(Protocol P): 7.177 minutes; HPLC 3.9 (Protocol O): HC Δ mass = 1180H-(C)_mc-#36 — 4.2 H-(C)_mcValCitPABC-#60 — 3.8 H-(C)_MalPeg3C2-#26 —3.8 H-(C)_mcValCitPABCAmPeg3C2-#36 — 3.5 H-A114C- — 1.9(C114)_mcValCitPABCAmPeg3C2-#36 H-A114C-(C114)_MalPeg6C2-#54 — 2H-(C)_MalPeg3C2-#60 — 4.2 H-(C)_MalPeg6C2-#60 — 4.1 H-(C)_MalPeg6C2-#41— 4 H-(C)-mc-#69 SEC (Protocol P): 7.192 minutes; HPLC 4.5 (Protocol O):HC Δ mass = 899 H-(C)_MalPeg3C2-#36 — 5.2 H-(C)_mcValCitPABCAmPeg6C2-#36— 4.2 H-(C)_MalPeg6C2-#36 — 5 H-(C)_mcValCitPABCAmPeg3C2-#41 — 4.4H-(C)-MalPeg3C2-#41 SEC (Protocol P): 7.317 minutes; HPLC 4.3 (ProtocolO): HC Δ mass = 1032 H-(C)_mcValCitPABCAmPeg6C2-#60 — 4.1H-A114C-(C114)_mc-#66 — 1.8 H-L398C + L443C- — 3.8 (C398 +C443)_mcValCitPABC-#54 H-K392C + L443C- — 3.8 (C392 +C443)_mcValCitPABC-#54 H-L443C-(C443)_mcValCitPABC-#54 SEC (Protocol P):8.827 minutes; HPLC 2 (Protocol O): HC Δ mass = 1344 H-L398C + V422C- —3.4 (C398 + C422)_mcValCitPABC-#54 H-(C)-mc-#44 SEC (Protocol P): 7.195minutes; HPLC 4.4 (Protocol O): HC Δ mass = 915 H-(C)-Mal-PEG3C2-#45 SEC(Protocol P): 7.364 minutes; HPLC 4.3 (Protocol O): HC Δ mass = 1017H-(C)_2AcAmPeg6C2-#66 — 4 H-(C)-Mal-PEG6C2-#45 SEC (Protocol P): 7.419minutes; HPLC 3.9 (Protocol O): HC Δ mass = 1154 H-(C)-mc-#79 SEC(Protocol P): 7.478 minutes; HPLC 4 (Protocol O): HC Δ mass = 946H-(C)-MalPeg3C2-#44 SEC (Protocol P): 7.247 minutes; HPLC 3.4 (ProtocolO): HC Δ mass = 1005 H-(C)-mcValCitPABC-#70 SEC (Protocol P): 7.466minutes; HPLC 3.7 (Protocol O): HC Δ mass = 1318 H-(C)-MalPeg6C2-#44 SEC(Protocol P): 7.237 minutes; HPLC 3.4 (Protocol O): HC Δ mass = 1135H-A114C-(C114)_mcValCitPABC-#69 — 2 H-(C)-mcValCitPABC-#79 SEC (ProtocolP): 7.635 minutes; HPLC 3.7 (Protocol O): HC Δ mass = 1349H-A114C-(C114)_mcValCitPABC-#79 — 1.84 H-(C)-mcValCitPABC-#44 SEC(Protocol P): 7.351 minutes; HPLC 4.2 (Protocol O): HC Δ mass = 1321H-A114C-(C114)_mcValCitPABC-#88 1.93 H-(C)-mcValCitPABC-#69 SEC(Protocol P): 7.254 minutes; HPLC 4.5 (Protocol O): HC Δ mass = 1305H-(C)_2AcAmCapValCitPABC-#66 — 3.3 H-A114C-(C114)_mcValCitPABC-#45 —1.92 H-A114C-(C114)_mcValCitPABC-#34 — 2 H-A114C-(C114)_mc-#45 — 1.95H-A114C-(C114)_mc-#70 — 2 H-(C)_mcValCitPABC-#112 SEC (Protocol P):7.083 minutes; HPLC 4.4 (Protocol O): HC Δ mass = 1291H-(C)-Mal-PEG6C2-#69 SEC (Protocol P): 7.350 minutes; HPLC 3.4 (ProtocolO): HC Δ mass = 1122 H-Q347C-(C347)_mcValCitPABC-#69 — 2H-Y373C-(C373)_mcValCitPABC-#69 — 1.6 H-E388C-(C388)_mcValCitPABC-#69 —2 H-N421C-(C421)_mcValCitPABC-#69 — 1.95 H-L443C-(C443)_mcValCitPABC-#69— 2 H-L443C-(C443)_mcValCitPABC-#79 — 2 H-A114C-(C114)_mcValCitPABC-#95— 2 H-A114C-(C114)_mcValCitPABC-#98 — 2 H-A114C-(C114)_MalPeg3C2-#69 — 2H-N297Q-(Q)_AmPeg6C2-#42 — 3.2 H-N297Q-(Q)_AmPeg6C2-#54 — 3.04H-N297Q-(Q)_AmPeg6C2-#47 — 3.16 H-N297Q-(Q)_AmPeg6C2-#36 — 3.36H-N297Q-(Q)_AmPeg6C2-#26 — 3.4 H-N297Q-(Q)_AmPeg6C2-#66 — 2.8H-L443C-(C443)_MalPeg6C2-#69 SEC (Protocol P): 7.012 minutes; HPLC 2(Protocol O): HC Δ mass = 1120 H-Q347C-(C347)_MalPeg6C2-#69 — 1.9H-E388C-(C388)_MalPeg6C2-#69 — 1.8 H-N421C-(C421)_MalPeg6C2-#69 — 1.8H-E380C-(C380)_MalPeg6C2-#69 — 1.8 H-L398C + L443C- — 3.9 (C398 +C443)_MalPeg6C2-#69 H-K392C + L443C- — 3.5 (C392 + C443)_MalPeg6C2-#69H-kA111C-(kC111)_MalPeg6C2-#69 — 3.7 H-kK183C-(kC183)_MalPeg6C2-#69 —2.1 H-kK207C-(kC207)_MalPeg6C2-#69 — 2.3H-A114C-(C114)_mcValCitPABC-#108 — 2 H-A114C-(C114)_mcValCitPABC-#84 —1.9 H-A114C-(C114)_mcValCitPABC-#226 — 1.8 H-A114C-(C114)_mc-#108 — 1.9H-A114C-(C114)_mcValCitPABC-#117 — 1.8 H-A114C-(C114)_mcValCitPABC-#115— 1.9 H-A114C-(C114)_MalPeg6C2-#98 — 1.9 IL13Ra2-AB08-v1010-hG1- — 3.9(C)_mcValCitPABC-#54 IL13Ra2-AB08-v1010-hG1-(C)_mc-#69 — 3.5IL13Ra2-AB08-v1010-hG1- — 3.5 (C)_MalPeg6C2-#69 IL13Ra2-AB08-v1010-hG1-— 4.4 (C)_mcValCitPABC-#69 H-A114C-(C114)_MalPeg6C2-0#118 — 1.9H-A114C-(C114)_mcValCitPABC- — 1.8 0#118 H-A114C-(C114)_mcValCitPABC-#80— 1.8 H-A114C-(C114)_mc-#117 — 1.9 H-A114C-(C114)_mcValCitPABC-#232 —1.8 H-A114C-(C114)_MalPeg6C2-#230 — 1.9 H-A114C-(C114)_MalPeg6C2-#117 —1.9 H-A114C-(C114)_mc-#115 — 2 H-A114C-(C114)_mv-#115 — 2H-A114C-(C114)_mb-#69 — 2 H-A114C-(C114)_mv-#69 — 2H-A114C-(C114)_mc-0#118 — 2 H-(C)_mc-#117 SEC (Protocol P): 7.797minutes; HPLC 3.5 (Protocol O): HC Δ mass = 937 H-(C)_MalPeg6C2-#117 SEC(Protocol P): 8.005 minutes; HPLC 3.56 (Protocol O): HC Δ mass = 1163H-(C)_mc-0#118 — 4.1 H-(C)_MalPeg6C2-0#118 SEC (Protocol P): NA; HPLC(Protocol O): HC 3.9 Δ mass = 1148 IL13Ra2-AB08-v1010-hG1-(C)_mc- — 40#118 IL13Ra2-AB08-v1010-hG1-(C)_mc- — 4.6 #226IL13Ra2-AB08-v1010-hG1-(C)_mc- — 3.3 #117 IL13Ra2-AB08-v1010-hG1- — 3.3(C)_MalPeg6C2-#117 IL13Ra2-AB08-v1010-hG1- — 2.9 (C)_MalPeg6C2-0#118H-A114C-(C114)_MalPeg6C2-#226 — 1.9 H-A114C-(C114)_mc-#172 — 1.9H-A114C-(C114)_mb-0#118 — 1.9 H-A114C-(C114)_me-0#118 — 2H-A114C-(C114)_mcValCitPABC-#134 — 1.9 H-A114C-(C114)_mc-#131 — 2H-A114C-(C114)_MalPeg6C2-#126 — 1.9 H-A114C-(C114)_MalPeg6C2-#123 — 1.7H-A114C-(C114)_mc-#126 — 2 H-A114C-(C114)_mv-0#118 — 2H-(C)_MalPeg6C2-#226 SEC (Protocol P): 7.501 minutes; HPLC 4.5 (ProtocolO): HC Δ mass = 1150 H-(C)_mc-#226 SEC (Protocol P): 7.418 minutes; HPLC4.5 (Protocol O): HC Δ mass = 927 IL13Ra2-AB08-v1010-hG1- — 4.2(C)_MalPeg6C2-#226 Notch-28-cG1-(C)_mc-0#118 — 4.6Notch-28-cG1-(C)_mc-#115 SEC (Protocol P): 7.015 minutes; HPLC 3.7(Protocol O): HC Δ mass = 911 Notch-28-cG1-(C)_MalPeg6C2-0#118 — 4.1Notch-28-cG1-(C)_me-0#118 SEC (Protocol P): 7.182 minutes; HPLC 3.9(Protocol O): HC Δ mass = 937 Notch-75-cG1-(C)_mc-0#118 — 3.3IL13Ra2-19F9-cG1- — 4.1 (C)_mcValCitPABC-#54 IL13Ra2-19F9-cG1- — 4.2(C)_mcValCitPABC-#112 Notch-28-cG1-(C)_mcValCitPABC- — 4.1 #112Notch-28-cG1-(C)_MalPeg6C2-#69 — 4.3 Notch-75-cG1-(C)_MalPeg6C2-#69 —3.8 H-(C)_m(H2O)c-0#118 SEC (Protocol P): 7.010 minutes; HPLC 4.1(Protocol O): HC Δ mass = 942 H-(C)_Mal(H2O)Peg6C2-0#118 SEC (ProtocolP): 6.964 minutes; HPLC 4 (Protocol O): HC Δ mass = 1166H-(C)_Mal(H2O)Peg6C2-#69 — 2.8 H-(C)_m(H2O)c-#69 — 3.6 H-(C)_me-0#118 —4.4 H-(C)_mv-0#118 — 4.4 H-(C)_mb-0#118 SEC (Protocol P): 7.032 minutes;HPLC 4.1 (Protocol O): HC Δ mass = 896 H-A114C-(C114)_MalC6-#54 — 1.9H-A114C-(C114)_mc-#231 — 1.7 H-A114C-(C114)_MalC6-0#118 — 2H-(C)_Mal(H2O)Peg6C2-#115 SEC (Protocol P): 6.936 minutes; HPLC 4.1(Protocol O): HC Δ mass = 1152 H-A114C-(C114)_mc-#158 — 2H-A114C-(C114)_mcValCitPABC-#231 — 1.7 H-(C)_m(H2O)c-#115 SEC (ProtocolP): 6.928 minutes; HPLC 3.7 (Protocol O): HC Δ mass = 930Notch-75-cG1-(C)_mc-#115 — 3.7 Notch-75-cG1-(C)_me-0#118 — 3.5Notch-75-cG1-(C)_MalPeg6C2-0#118 — 3.8 H-A114C-(C114)_mc-#237 — 2H-A114C-(C114)_mc-#145 — 2 H-A114C-(C114)_MalPeg6C2-#145 — 2H-A114C-(C114)_mc-#162 — 1.9 H-A114C-(C114)_MalC6Am-#151 — 1.9Notch-28-cG1-(C)_m(H2O)c-0#118 — 3.7 Notch-75-cG1-(C)_m(H2O)c-0#118 — 3H-(kK188)_COPeg2C2ValCitPABC- — 2 #54 IL13Ra2-AB08-v1010-hG1- SEC(Protocol P): 7.766 minutes; HPLC 3.5 (C)_Mal(H2O)Peg6C2-0#118 (ProtocolO): HC Δ mass = 1164 IL13Ra2-AB08-v1010-hG1- — 3.9(C)_Mal(H2O)Peg6C2-#115 IL13Ra2-AB08-v1010-hG1-(C)_mc- SEC (Protocol P):7.813 minutes; HPLC 4.3 #115 (Protocol O): HC Δ mass = 911IL13Ra2-AB08-v1010-hG1- — 3.3 (C)_m(H2O)c-0#118 H-(C)_mcValCitPABC-0#118— 4.5 IL13Ra2-AB08-v1010-hG1- SEC (Protocol P): 7.783 minutes; HPLC 3.8(C)_m(H2O)c-#115 (Protocol O): HC Δ mass = 930H-A114C-(C114)_mcValCitPABC-#154 — 1.9 H-A114C-(C114)_MalC6Am-#153 — 2IL13Ra2-AB08-v1010- — 4.3 Q347C + kK183C-hG1- (C347 +kC183)_mcValCitPABC-#54 IL13Ra2-AB08-v1010-Q347C-hG1- — 2.1(C347)_mcValCitPABC-#54 IL13Ra2-AB08-v1010-hG1- — 2(kK188)_COPeg2C2AmPeg2C2-#69 IL13Ra2-AB08-v1010-hG1- — 1.9(kK188)_COPeg2C2ValCitPABC-#54 IL13Ra2-AB08-v1010-L443C-hG1- — 2.1(C443)_mcValCitPABC-#54 IL13Ra2-AB08-v1010-K392C + L443C- — 3.7hG1-(C392 + C443)_mcValCitPABC-#54 IL13Ra2-AB08-v1010-L443C + kK183C- —4 hG1-(C443 + kC183)_mcValCitPABC- #54 H-(C)_mcValCitPABC-#98 SEC(Protocol P): 7.232 minutes; HPLC 4.2 (Protocol O): HC Δ mass = 1371H-A114C-(C114)_mcValCitPABC-#246 — 1.9 H-H435A-(C)_mcValCitPABC-#54 — 4H-M428L + N434S-(C)_mcValCitPABC- — 4.2 #70 H-M428L +N434S-(C)_mcValCitPABC- — 4 #54 H-E388C + N421C- — 3.6 (C388 +C421)_mcValCitPABC-#54 H-Q347C + K392C- — 3.9 (C347 +C392)_mcValCitPABC-#54 H-L443C + kK183C- — 3.7 (C443 +kC183)_mcValCitPABC-#54 H-Q347C + kK183C- SEC (Protocol P): 8.278minutes; HPLC 3.7 (C347 + kC183)_mcValCitPABC-#54 (Protocol O): HC Δmass = 1339 H-Q347C-(C347)_mcValCitPABC-#54 — 1.9 H-K392C +L443C-(C392 + C443)_mc- — 4 #115 H-E388C + N421C-(C388 + C421)_mc- — 3.8#115 H-Q347C + K392C-(C347 + C392)_mc- — 4 #115 H-L443C + kK183C-(C443 +kC183)_mc- — 3.8 #115 H-Q347C + kK183C-(C347 + kC183)_mc- — 3.8 #115H-Q347C-(C347)_mc-#115 — 2 H-kK183C-(kC183)_mcValCitPABC- — 1.9 #54H-E388C-(C388)_mcValCitPABC-#54 — 2 H-kK183C-(kC183)_mc-#115 — 1.8H-E388C-(C388)_mc-#115 SEC (Protocol P): 7.364 minutes; HPLC 2 (ProtocolO): HC Δ mass = 914 H-L443C-(C443)_mc-#115 — 2H-N421C-(C421)_mcValCitPABC-#54 — 2 H-N421C-(C421)_mc-#115 — 2H-A114C-(C114)_mcGly-#201 — 1.9

TABLE 20 IC₅₀ values for selected compounds (cytotoxic peptides) of theinvention MDA-MB- BT474 361-DYT2 GMEAN N87GMEAN GMEAN IC₅₀ Example #IC₅₀ (nM) IC₅₀ (nM) (nM) #26 0.368 0.543 1.045 #30 0.682 6.709 1.853 #340.211 1.95 1.119 #36 0.499 1.205 1.111 #41 29.666 33.21 51.784 #42 0.1250.327 0.195 #44 7.119 14.61 >16.401 #45 0.15 0.385 0.415 #47 <0.244<0.256 0.317 #51 <0.599 3.658 — #54 <0.133 <0.221 0.206 #56 0.316 1.2560.766 #60 0.524 1.245 0.957 #66 0.244 0.463 0.334 #69 80.191 65.97940.988 #70 0.179 0.327 0.225 #75 >100.000 >100.000 >100.000 #79 0.0790.137 0.129 #80 20.346 28.204 32.846 #84 0.246 0.426 0.686 #115 31.49350.302 19.870 #117 0.096 0.103 0.118 #118 100.000 100.000 100.000 #1230.125 0.089 0.129 #126 0.315 0.375 0.454 #130 0.050 0.076 0.039 #1310.072 0.185 0.081 #134 0.108 0.115 0.134 #140 — — — #141 3.367 3.018 —#142 0.279 0.259 — #143 — — — #144 0.172 0.182 0.174 #145 0.185 0.1670.229 #146 0.435 0.195 0.387 #147 0.456 0.144 0.421 #148 0.429 0.2190.502 #149 0.417 0.250 0.428 #151 84.867 61.953 84.599 #153 98.16047.274 91.350 #154 0.193 0.572 0.198 #155 0.323 0.875 0.318 #158 0.0820.115 0.100 #159 0.070 0.075 0.074 #162 31.448 21.610 27.824 #163100.000 72.703 99.433 #172 0.057 0.144 0.086 #173 0.088 0.099 0.067 #1780.968 1.262 0.911 #180 0.159 0.117 0.113 #182 0.153 0.148 0.122 #1842.478 5.098 3.427 #186 — — — #188 0.250 0.283 0.404 #190 0.134 0.0660.095 #192 0.262 0.360 0.408 #194 0.134 0.212 0.198 #200 0.048 0.0290.017 #201 0.144 0.150 0.121 #207 0.219 0.626 0.260 #208 0.418 0.3790.336 #209 0.067 0.067 0.058 #217 — — — #219 — — — #220 35.163 100.000100.000 #221 32.402 87.857 65.401 #222 0.158 0.352 0.272 #223 7.58913.026 10.863 #224 0.383 1.563 0.998 #225 3.449 10.524 7.575 #226 0.1180.478 0.106 #227 11.008 18.975 12.899 #228 0.105 0.090 0.078 #229 18.37216.566 10.218 #230 100.000 89.133 70.236 #231 3.706 15.127 22.855 #2320.071 0.194 0.095 #233 1.074 8.413 5.042 #234 0.684 0.756 2.004 #2350.852 1.320 1.278 #236 0.020 0.023 0.010 #237 0.162 0.217 0.278 #2380.139 0.077 0.084 #239 — — — #240 11.710 19.930 23.480 #241 0.364 0.3880.494 #242 32.330 41.329 34.529 #243 1.252 1.301 1.284 #244 73.123100.000 100.000 #245 11.793 33.037 31.856 #246 3.159 10.828 5.430 #2471.007 2.061 1.334 #257 — — —

TABLE 21A IC₅₀ values for selected conjugates of the invention BT474HCC1954 N87 IC50 of IC50 of IC50 of IC₅₀ Antibody IC₅₀ Antibody IC₅₀Antibody ADC-Linker-Payload # (nM) (ng/mL) (nM) (ng/mL) (nM) (ng/mL)H-(C)_MalPeg3C2-#41 0.725 25.592 0.465 16.617 4.02 175.448H-(C)_MalPeg6C2-#42 0.502 19.855 0.604 24.783 >14.090 >6681.150H-(C)_mc-#44 3.553 121.414 14.464 493.077 >841.360 >29495.311H-(C)_MalPeg3C2-#44 2.603 114.847 5.113 225.594 >440.881 >26346.532H-(C)_MalPeg6C2-#44 1.318 58.155 1.466 64.663 98.174 4873.851H-(C)_mcValCitPABC-#44 0.188 6.717 0.155 5.329 0.781 28.146H-(C)_Mal-PEG3C2-#45 2.886 103.762 1.513 52.791 >740.001 >27967.742H-(C)_Mal-PEG6C2-#45 1.274 49.903 1.423 54.715 111.434 9072.131H-(C)_mcValCitPABC-#45 0.258 9.997 0.204 7.988 0.417 16.737 H-(C)_mc-#540.436 19.821 0.992 45.072 2.45 138.026 H-(C)_Mal-PEG6C2-#69 1.9385147.54 0.356 12.995 5.743 2427.639 H-(C)_mcValCitPABC-#69 0.18 7.170.073 2.878 <0.185 <8.946 H-(C)_mcValCitPABC-#70 0.133 4.522 0.078 2.610.249 8.722 H-(C)_mc-#79 0.483 18.097 0.654 24.543 7.576 297.254H-(C)_mcValCitPABC-#79 0.152 6.634 0.127 5.682 0.469 21.134H-(C)_mc-#115 0.272 — 0.109 — 0.841 — H-A114C-(C114)_mc-#51 41.768 — —17.297 — H-A114C-(C114)_mc-#47 3.269 — — 8.216 — H-A114C-(C114)_mc-#544.294 — — 7.195 — H-A114C- 0.493 — 0.31 — 0.696 —(C114)_mcValCitPABC-#47 H-A114C- 0.174 — 0.17 — 0.189 —(C114)_mcValCitPABC-#54 H-A114C- 2.548 — 28.2 — 4.314 —(C114)_mcValCitPABC-#26 H-A114C-(C114)_mc-#26 >60.648 — >1000.00— >980.026 — H-A114C- 2.007 — 26.18 — 13.579 — (C114)_mcValCitPABC-#36H-A114C- 0.283 0.16 — 0.524 — (C114)_mcValCitPABC-#42H-A114C-(C114)_mc-#42 0.81 — 1.54 — 44.164 — H-(C)-mcValCitPABC-#540.292 — 0.27 — 0.345 — H- 15.134 — 14.33 — 41.016 —(C)_mcValCitPABCAmPeg3C2- #54 H- 1.898 — 1.4 — 11.71 —(C)_mcValCitPABCAmPeg6C2- #54 H-(C)_mc-#47 4.429 — 3.52 — 20.007 —H-(C)_MalPeg3C2-#54 2.181 — 1.54 — >41.711 — H-(C)_mc-#54 3.565 — 6.28 —48.566 — H- 5.228 — >1000.00 — >543.852 — (C)_mcValCitPABCAmPeg3C2- #47H-(C)_MalPeg3C2-#47 1.467 — 1.29 — 16.856 — H- 1.587 — 4.95 — >1000.000— (C)_mcValCitPABCAmPeg3C2- #42 H-(C)_mc-#41 0.506 — 0.68 — 7.543 — H-11.211 — >1000.00 — >1000.000 — (C)_mcValCitPABCAmPeg3C2- #26 H- 0.935 —2.46 — 14.283 — (C)_mcValCitPABCAmPeg6C2- #47 H-(C)_MalPeg3C2-#42 0.517— 0.51 — 5.479 — H- 10.992 — >1000.00 — >1000.000 —(C)_mcValCitPABCAmPeg6C2- #26 H- 1.819 — 1.97 — 75.643 —(C)_mcValCitPABCAmPeg6C2- #42 H-(C)_MalPeg6C2-#54 2.108 — 1.02 — >56.928— H-(C)_MalPeg6C2-#47 1.637 — 1.42 — 31.762 — H-(C)_MalPeg6C2-#26 6.385— 9.55 — >817.859 — H-(C)-MalPeg6C2-#42 0.518 — 0.55 — >7.993 —H-(C)_mc-#36 >1000.000 — >1000.00 — >1000.000 — H-(C)_mcValCitPABC-#600.835 — 6.45 — 14.917 — H-(C)_MalPeg3C2-#26 11.506 — 9.43 — >1000.000 —H- >1000.000 — >1000.00 — >1000.000 — (C)_mcValCitPABCAmPeg3C2- #36H-A114C- >1000.000 — >1000.00 — >325.714 — (C114)_mcValCitPABCAmPeg3C2-#36 H-A114C-(C114)_MalPeg6C2- 1.228 — 2.01 — 133.426 — #54H-(C)_MalPeg3C2-#60 >1000.000 — >1000.00 — >1000.000 —H-(C)_MalPeg6C2-#60 >1000.000 — >1000.00 — >1000.000 —H-(C)_MalPeg6C2-#41 1.166 — 0.36 — 5.882 — H-(C)-mc-#69 0.427 — 0.47 —3.05 — H-(C)_MalPeg3C2-#36 720.826 — >1000.00 — >1000.000 — H- >1000.000— >1000.00 — >1000.000 — (C)_mcValCitPABCAmPeg6C2- #36H-(C)_MalPeg6C2-#36 878.903 — 159.1 — >1000.000 — H- 2.363 — 2.28 —18.728 — (C)_mcValCitPABCAmPeg3C2- #41 H-(C)-MalPeg3C2-#41 0.725 — 0.54— 4.004 — H- 979.982 — >1000.00 — 392.905 — (C)_mcValCitPABCAmPeg6C2-#60 H-A114C-(C114)_mc-#66 17.235 — >1000.00 — >1000.000 — H-L398C +L443C- 0.249 — 0.27 — 0.678 — (C398 + C443)_mcValCitPABC- #54 H-K392C +L443C- <0.195 — 0.42 — <0.254 — (C392 + C443)_mcValCitPABC- #54 H-L443C-<0.130 — 0.32 — <0.267 — (C443)_mcValCitPABC-#54 H-L398C + V422C- 0.387— 0.27 — 0.504 — (C398 + C422)_mcValCitPABC- #54 H-(C)-mc-#44 3.553— >507.23 — >878.489 — H-(C)-Mal-PEG3C2-#45 2.886 — 68.41 — >834.717 —H-(C)_2AcAmPeg6C2-#66 703.419 — >1000.00 — >1000.000 —H-(C)-Mal-PEG6C2-#45 1.274 — 2.74 — >268.047 — H-(C)-mc-#79 0.483 — 0.65— 7.576 — H-(C)-MalPeg3C2-#44 2.603 — 5.11 — >440.881 —H-(C)-mcValCitPABC-#70 0.188 — 0.09 — <0.179 — H-(C)-MalPeg6C2-#44 1.318— 1.47 — 98.174 — H-A114C- 0.174 — 0.06 — 0.207 —(C114)_mcValCitPABC-#69 H-(C)-mcValCitPABC-#79 0.152 — 0.15 — 0.469 —H-A114C- 0.124 — 0.12 — 0.386 — (C114)_mcValCitPABC-#79H-(C)-mcValCitPABC-#44 0.252 — 0.18 — 0.732 — H-A114C- 8.127 — >1000.00— 62.825 — (C114)_mcValCitPABC-#88 H-(C)-mcValCitPABC-#69 0.133 — 0.1 —0.249 — H-(C)_2AcAmCapValCitPABC- 0.436 — 0.99 — 2.45 — #66 H-A114C-0.217 — 0.2 — 0.496 — (C114)_mcValCitPABC-#45 H-A114C- 3.724 — >1000.00— 18.422 — (C114)_mcValCitPABC-#34 H-A114C-(C114)_mc-#45 6.431— >1000.00 — 148.852 — H-A114C-(C114)_mc-#70 0.349 — 0.62 — 7.208 —H-(C)_mcValCitPABC-#112 0.226 — 0.24 — 0.469 — H-(C)-Mal-PEG6C2-#690.453 — 0.54 — 1.8 — H-Q347C- 0.368 — 0.06 — 0.22 —(C347)_mcValCitPABC-#69 H-Y373C- 0.359 — 0.06 — 0.295 —(C373)_mcValCitPABC-#69 H-E388C- 0.427 — 0.06 — 0.314 —(C388)_mcValCitPABC-#69 H-N421C- 0.434 — 0.09 — 0.244 —(C421)_mcValCitPABC-#69 H-L443C- 0.239 — 0.05 — 0.272 —(C443)_mcValCitPABC-#69 H-L443C- 0.3 — 0.15 — 0.412 —(C443)_mcValCitPABC-#79 H-A114C- 0.381 — 0.36 — 0.852 —(C114)_mcValCitPABC-#95 H-A114C- 0.171 — 0.24 — 0.258 —(C114)_mcValCitPABC-#98 H-A114C-(C114)_MalPeg3C2- 0.221 — 0.58 — 1.589 —#69 H-N297Q-(Q)_AmPeg6C2-#42 0.466 — 0.36 — 5.42 —H-N297Q-(Q)_AmPeg6C2-#54 0.557 — 0.37 — 6.899 — H-N297Q-(Q)_AmPeg6C2-#470.346 — 0.43 — 4.337 — H-N297Q-(Q)_AmPeg6C2-#36 3.003 — >1000.00 —284.267 — H-N297Q-(Q)_AmPeg6C2-#26 0.991 — 1.07 — 35.331 —H-N297Q-(Q)_AmPeg6C2-#66 13.812 — >1000.00 — >1000.000 —H-L443C-(C443)_MalPeg6C2- 0.251 — 0.25 — 1.989 — #69H-Q347C-(C347)_MalPeg6C2- 0.267 — 0.3 — 0.887 — #69H-E388C-(C388)_MalPeg6C2- 0.382 — 0.46 — 3.035 — #69H-N421C-(C421)_MalPeg6C2- 0.35 — 0.45 — 1.329 — #69H-E380C-(C380)_MalPeg6C2- 0.482 — 0.49 — 5.588 — #69 H-L398C + L443C-0.226 — 0.3 — 1.346 — (C398 + C443)_MalPeg6C2-#69 H-K392C + L443C- 0.268— 0.31 — 1.63 — (C392 + C443)_MalPeg6C2-#69 H-kA111C- 0.297 — 0.34 —1.635 — (kC111)_MalPeg6C2-#69 H-kK183C- 0.257 — 0.5 — 2.23 —(kC183)_MalPeg6C2-#69 H-kK207C- 0.252 — 0.41 — 1.744 —(kC207)_MalPeg6C2-#69 H-A114C- 0.212 — 0.12 — 0.777 —(C114)_mcValCitPABC-#108 H-A114C- 0.627 — 12.2 — 1.733 —(C114)_mcValCitPABC-#84 H-A114C- 0.2 — 0.1 — 0.239 —(C114)_mcValCitPABC-#226 H-A114C-(C114)_mc-#108 >1000.000 — >1000.00 —113.889 — H-A114C- 0.242 — 0.17 — 0.239 — (C114)_mcValCitPABC-#117H-A114C- 0.202 — 0.2 — 0.211 — (C114)_mcValCitPABC-#115H-A114C-(C114)_MalPeg6C2- 0.576 — 0.47 — 1.46 — #98H-A114C-(C114)_MalPeg6C2- 0.257 — 0.17 — 0.505 — 0#118 H-A114C- 0.251 —0.24 — 0.398 — (C114)_mcValCitPABC-0#118 H-A114C- 0.341 — 0.31 — 0.887 —(C114)_mcValCitPABC-#80 H-A114C-(C114)_mc-#117 0.197 — 0.14 — 0.465 —H-A114C- 0.376 — 1.31 — 1.367 — (C114)_mcValCitPABC-#232H-A114C-(C114)_MalPeg6C2- 0.504 — 0.85 — 3.179 — #230H-A114C-(C114)_MalPeg6C2- 0.335 — 0.21 — 0.792 — #117H-A114C-(C114)_mc-#115 0.243 — 0.23 — 0.45 — H-A114C-(C114)_mv-#115 0.21— 0.15 — 0.65 — H-A114C-(C114)_mb-#69 0.256 — 0.43 — 2.137 —H-A114C-(C114)_mv-#69 0.215 — 0.27 — 1.043 — H-A114C-(C114)_mc-0#1180.151 — 0.1 — 0.342 — H-(C)_mc-#117 0.162 — 0.06 — 0.314 —H-(C)_MalPeg6C2-#117 0.283 — 0.07 — 0.515 — H-(C)_mc-0#118 0.18 — <0.10— 0.303 — H-(C)_MalPeg6C2-0#118 0.269 — 0.15 — 0.499 —H-A114C-(C114)_MalPeg6C2- 0.28 — 0.22 — 0.685 — #226H-A114C-(C114)_mc-#172 0.296 — 0.41 — 0.694 — H-A114C-(C114)_mb-0#1180.318 — 0.33 — 0.709 — H-A114C-(C114)_me-0#118 0.256 — 0.33 — 0.64 —H-A114C- 0.301 — 0.34 — 0.501 — (C114)_mcValCitPABC-#134H-A114C-(C114)_mc-#131 0.357 — 0.76 — 1.614 — H-A114C-(C114)_MalPeg6C2-0.284 — 0.36 — 1.377 — #126 H-A114C-(C114)_MalPeg6C2- 0.362 — 0.34 —1.867 — #123 H-A114C-(C114)_mc-#126 0.319 — 0.49 — 3.294 —H-A114C-(C114)_mv-0#118 0.209 — 0.25 — 0.719 — H-(C)_MalPeg6C2-#2260.575 — 0.22 — 1.126 — H-(C)_mc-#226 0.359 — 0.18 — 0.69 —H-(C)_m(H2O)c-0#118 0.26 — 0.11 — 0.448 — H-(C)_Mal(H2O)Peg6C2-0#1180.482 — 0.19 — 0.9 — H-(C)_Mal(H2O)Peg6C2-#69 0.832 — 0.51 — 5.769 —H-(C)_m(H2O)c-#69 0.418 — 0.28 — 1.529 — H-(C)_me-0#118 0.186 — 0.11 —0.218 — H-(C)_mv-0#118 0.201 — 0.14 — 0.265 — H-(C)_mb-0#118 0.222 —0.13 — 0.267 — H-A114C-(C114)_MalC6-#54 0.662 — 5.11 — 8.003 —H-A114C-(C114)_mc-#231 >1000.000 — >1000.00 — >1000 —H-A114C-(C114)_MalC6-0#118 0.976 — 113 — 15.407 —H-(C)_Mal(H2O)Peg6C2-#115 1.06 — 0.28 — 3.439 — H-A114C-(C114)_mc-#1580.247 — 0.35 — 0.739 — H-A114C- 1.178 — 24.41 — 13.447 —(C114)_mcValCitPABC-#231 H-(C)_m(H2O)c-#115 0.393 — 0.17 — 0.498 —H-A114C-(C114)_mc-#237 0.97 — 0.68 — 27.907 — H-A114C-(C114)_mc-#1454.681 — 585.59 — 643.391 — H-A114C-(C114)_MalPeg6C2- 12.856 — 190.59 —89.125 — #145 H-A114C-(C114)_mc-#162 0.377 — 0.15 — 1.144 —H-A114C-(C114)_MalC6Am- 0.42 — 0.1 — 0.694 — #151 H- — — —(kK188)_COPeg2C2ValCitPABC- #54 H-(C)_mcValCitPABC-0#118 0.227 — 0.14 —0.182 — H-A114C- 0.323 — 0.32 — 0.363 — (C114)_mcValCitPABC-#154H-A114C-(C114)_MalC6Am- 0.377 — 0.27 — 0.34 — #153H-(C)_mcValCitPABC-#98 0.211 — 0.14 — 0.162 — H-A114C- 0.357 — 0.65 —3.197 — (C114)_mcValCitPABC-#246 H-H435A-(C)_mcValCitPABC- 0.358 — 0.17— 0.237 — #54 H-M428L + N434S- 0.322 — 0.1 — 0.114 —(C)_mcValCitPABC-#70 H-M428L + N434S- 0.354 — — 0.217 —(C)_mcValCitPABC-#54 H-E388C + N421C- 1.38 — 0.99 — 0.855 — (C388 +C421)_mcValCitPABC- #54 H-Q347C + K392C- 0.276 — 0.29 — 0.147 — (C347 +C392)_mcValCitPABC- #54 H-L443C + kK183C- <0.129 — 0.37 — <0.111 —(C443 + kC183)_mcValCitPABC- #54 H-Q347C + kK183C- 0.146 — 0.25 — 0.08 —(C347 + kC183)_mcValCitPABC- #54 H-Q347C- 0.153 — 0.33 — 0.111 —(C347)_mcValCitPABC-#54 H-K392C + L443C- 0.323 — 0.1 — 0.304 — (C392 +C443)_mc-#115 H-E388C + N421C- 1.251 — 0.42 — 0.997 — (C388 +C421)_mc-#115 H-Q347C + K392C- 0.342 — 0.1 — 0.219 — (C347 +C392)_mc-#115 H-L443C + kK183C- 0.319 — 0.1 — 0.268 — (C443 +kC183)_mc-#115 H-Q347C + kK183C- 0.347 — 0.1 — 0.403 — (C347 +kC183)_mc-#115 H-Q347C-(C347)_mc-#115 0.272 — 0.18 — 0.278 — H-kK183C-0.287 — 0.34 — 0.194 — (kC183)_mcValCitPABC-#54 H-E388C- 0.098 — 0.38 —0.084 — (C388)_mcValCitPABC-#54 H-kK183C-(kC183)_mc-#115 0.28 — 0.27 —0.269 — H-E388C-(C388)_mc-#115 0.302 — 0.15 — 0.301 —H-L443C-(C443)_mc-#115 0.222 — 0.1 — 0.259 — H-N421C- <0.051 — 0.42 —<0.051 — (C421)_mcValCitPABC-#54 H-N421C-(C421)_mc-#115 0.312 — 0.23 —0.306 — H-A114C-(C114)_mcGly-#201 0.321 — — —

TABLE 21B IC₅₀ values for selected conjugates of the invention DYT2MDA-MB-468 IC50 of IC50 of IC₅₀ Antibody IC₅₀ AntibodyADC-Linker-Payload # (nM) (ng/mL) (nM) (ng/mL)H-(C)_MalPeg3C2-#41 >69.685 >17528.581 — >35714.286 H-(C)_MalPeg6C2-#4233.396 5455.61 >629.281 >25857.971H-(C)_mc-#44 >1000.000 >34090.909 >1000.000 >34090.909H-(C)_MalPeg3C2-#44 >1000.000 >44117.647 >1000.000 >44117.647H-(C)_MalPeg6C2-#44 >1000.000 >44117.647 >1000.000 >44117.647H-(C)_mcValCitPABC-#44 0.203 7.246 >1000.000 >35714.286H-(C)_Mal-PEG3C2-#45 >1000.000 >34883.721 >1000.000 >34883.721H-(C)_Mal-PEG6C2-#45 >1000.000 >38461.538 >1000.000 >38461.538H-(C)_mcValCitPABC-#45 0.371 14.304 613.294 24435.914H-(C)_mc-#54 >1000.000 >45454.545 >1000.000 >45454.545H-(C)_Mal-PEG6C2-#69 >467.163 >29849.279 >1000.000 >35714.286H-(C)_mcValCitPABC-#69 0.156 7.54 547.953 21860.354H-(C)_mcValCitPABC-#70 0.098 3.332 >1000.000 >33333.333H-(C)_mc-#79 >1000.000 >37500.000 978.508 36694.065H-(C)_mcValCitPABC-#79 0.212 9.528 351.392 15383.462 H-(C)_mc-#115 0.21— — — H-A114C-(C114)_mc-#51 >1000.000 — >1000.000 —H-A114C-(C114)_mc-#47 >1000.000 — >1000.000 —H-A114C-(C114)_mc-#54 >1000.000 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#47 383.667 — 445.014 —H-A114C-(C114)_mcValCitPABC-#54 0.372 — 362.213 —H-A114C-(C114)_mcValCitPABC-#26 >1000.000 — >930.555 —H-A114C-(C114)_mc-#26 >1000.000 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#36 >927.422 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#42 0.436 — 530.596 —H-A114C-(C114)_mc-#42 >727.245 — 567.735 — H-(C)-mcValCitPABC-#54 0.275— 471.905 — H-(C)_mcValCitPABCAmPeg3C2-#54 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg6C2-#54 >1000.000 — >1000.000 —H-(C)_mc-#47 >1000.000 — >1000.000 — H-(C)_MalPeg3C2-#54 >1651.007— >1651.007 — H-(C)_mc-#54 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg3C2-#47 >1000.000 — >1000.000 —H-(C)_MalPeg3C2-#47 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg3C2-#42 >1000.000 — >1000.000 —H-(C)_mc-#41 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg3C2-#26 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg6C2-#47 >1000.000 — >1000.000 —H-(C)_MalPeg3C2-#42 9.675 — 358.435 —H-(C)_mcValCitPABCAmPeg6C2-#26 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg6C2-#42 >1000.000 — >1000.000 —H-(C)_MalPeg6C2-#54 >1731.544 — >1731.544 —H-(C)_MalPeg6C2-#47 >1651.007 — >1651.007 —H-(C)_MalPeg6C2-#26 >1000.000 — >1000.000 — H-(C)-MalPeg6C2-#42 5.705— >642.029 — H-(C)_mc-#36 >1000.000 — >1000.000 —H-(C)_mcValCitPABC-#60 >699.241 — >544.495 —H-(C)_MalPeg3C2-#26 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg3C2-#36 >1000.000 — >1000.000 —H-A114C- >1000.000 — >1000.000 — (C114)_mcValCitPABCAmPeg3C2-#36H-A114C-(C114)_MalPeg6C2-#54 >1000.000 — >1000.000 —H-(C)_MalPeg3C2-#60 >1000.000 — >1000.000 —H-(C)_MalPeg6C2-#60 >1000.000 — >1000.000 —H-(C)_MalPeg6C2-#41 >1000.000 — >1000.000 — H-(C)-mc-#69 >71.831— >899.249 — H-(C)_MalPeg3C2-#36 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg6C2-#36 >1000.000 — >1000.000 —H-(C)_MalPeg6C2-#36 >1000.000 — >1000.000 —H-(C)_mcValCitPABCAmPeg3C2-#41 >1000.000 — >1000.000 —H-(C)-MalPeg3C2-#41 >69.685 — >1000.000 —H-(C)_mcValCitPABCAmPeg6C2-#60 >1000.000 — >1000.000 —H-A114C-(C114)_mc-#66 >1000.000 — >1000.000 — H-L398C + L443C- 0.463 —801.354 — (C398 + C443)_mcValCitPABC-#54 H-K392C + L443C- <0.171 —565.01 — (C392 + C443)_mcValCitPABC-#54 H-L443C-(C443)_mcValCitPABC-#540.371 — 500.958 — H-L398C + V422C- 0.48 — 610.884 — (C398 +C422)_mcValCitPABC-#54 H-(C)-mc-#44 >1000.000 — >1000.000 —H-(C)-Mal-PEG3C2-#45 >1000.000 — >1000.000 —H-(C)_2AcAmPeg6C2-#66 >1000.000 — >1000.000 —H-(C)-Mal-PEG6C2-#45 >1000.000 — >1000.000 — H-(C)-mc-#79 >1000.000 —978.508 — H-(C)-MalPeg3C2-#44 >1000.000 — >1000.000 —H-(C)-mcValCitPABC-#70 0.116 — 547.953 — H-(C)-MalPeg6C2-#44 >1000.000— >1000.000 — H-A114C-(C114)_mcValCitPABC-#69 0.083 — >1000.000 —H-(C)-mcValCitPABC-#79 0.212 — 351.392 — H-A114C-(C114)_mcValCitPABC-#790.199 — 472.593 — H-(C)-mcValCitPABC-#44 0.248 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#88 >1000.000 — >1000.000 —H-(C)-mcValCitPABC-#69 0.098 — >1000.000 —H-(C)_2AcAmCapValCitPABC-#66 >1000.000 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#45 2.37 — >968.025 —H-A114C-(C114)_mcValCitPABC-#34 >1000.000 — >1000.000 —H-A114C-(C114)_mc-#45 >1000.000 — >1000.000 —H-A114C-(C114)_mc-#70 >1000.000 — >1000.000 — H-(C)_mcValCitPABC-#1120.185 — >563.999 — H-(C)-Mal-PEG6C2-#69 0.963 — 748.275 —H-Q347C-(C347)_mcValCitPABC-#69 0.094 — >1000.000 —H-Y373C-(C373)_mcValCitPABC-#69 0.156 — >1000.000 —H-E388C-(C388)_mcValCitPABC-#69 0.117 — >1000.000 —H-N421C-(C421)_mcValCitPABC-#69 0.162 — >1000.000 —H-L443C-(C443)_mcValCitPABC-#69 0.1 — >1000.000 —H-L443C-(C443)_mcValCitPABC-#79 0.303 — 370.53 —H-A114C-(C114)_mcValCitPABC-#95 61.8 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#98 0.218 — 609.904 —H-A114C-(C114)_MalPeg3C2-#69 >1000.000 — >1000.000 —H-N297Q-(Q)_AmPeg6C2-#42 >1000.000 — >1000.000 —H-N297Q-(Q)_AmPeg6C2-#54 >1000.000 — >1000.000 —H-N297Q-(Q)_AmPeg6C2-#47 >1000.000 — >1000.000 —H-N297Q-(Q)_AmPeg6C2-#36 >1000.000 — >1000.000 —H-N297Q-(Q)_AmPeg6C2-#26 >1000.000 — >1000.000 —H-N297Q-(Q)_AmPeg6C2-#66 >1000.000 — >1000.000 —H-L443C-(C443)_MalPeg6C2-#69 >1000.000 — 758.157 —H-Q347C-(C347)_MalPeg6C2-#69 1.752 — 832.08 —H-E388C-(C388)_MalPeg6C2-#69 6.883 — >973.529 —H-N421C-(C421)_MalPeg6C2-#69 1.027 — 472.466 —H-E380C-(C380)_MalPeg6C2-#69 >65.641 — 873.254 — H-L398C + L443C- 0.827— 846.418 — (C398 + C443)_MalPeg6C2-#69 H-K392C + L443C- >32.438 —804.407 — (C392 + C443)_MalPeg6C2-#69 H-kA111C-(kC111)_MalPeg6C2-#690.423 — 740.791 — H-kK183C-(kC183)_MalPeg6C2-#69 >1000.000 — 749.154 —H-kK207C-(kC207)_MalPeg6C2-#69 >138.618 — 586.857 —H-A114C-(C114)_mcValCitPABC-#108 >1000.000 — 873.831 —H-A114C-(C114)_mcValCitPABC-#84 976.796 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#226 0.101 — 385.851 —H-A114C-(C114)_mc-#108 >1000.000 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#117 0.107 — 469.882 —H-A114C-(C114)_mcValCitPABC-#115 0.142 — 989.147 —H-A114C-(C114)_MalPeg6C2-#98 355.331 — >1000.000 —H-A114C-(C114)_MalPeg6C2-0#118 0.126 — >865.455 —H-A114C-(C114)_mcValCitPABC-0#118 0.215 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#80 0.432 — >1000.000 —H-A114C-(C114)_mc-#117 0.107 — >414.892 —H-A114C-(C114)_mcValCitPABC-#232 38.422 — 959.259 —H-A114C-(C114)_MalPeg6C2-#230 >1000.000 — >1000.000 —H-A114C-(C114)_MalPeg6C2-#117 0.179 — >1000.000 — H-A114C-(C114)_mc-#1150.238 — >699.755 — H-A114C-(C114)_mv-#115 0.322 — >668.891 —H-A114C-(C114)_mb-#69 >1000.000 — >1000.000 —H-A114C-(C114)_mv-#69 >1000.000 — >1000.000 — H-A114C-(C114)_mc-0#1180.098 — 432.816 — H-(C)_mc-#117 <0.093 — 194.684 — H-(C)_MalPeg6C2-#117<0.080 — 361.061 — H-(C)_mc-0#118 <0.073 — 541.542 —H-(C)_MalPeg6C2-0#118 0.074 — 465.455 — H-A114C-(C114)_MalPeg6C2-#2260.513 — 574.794 — H-A114C-(C114)_mc-#172 1.48 — 500.864 —H-A114C-(C114)_mb-0#118 0.208 — 506.604 — H-A114C-(C114)_me-0#118 0.236— 903.571 — H-A114C-(C114)_mcValCitPABC-#134 1.434 — 648.066 —H-A114C-(C114)_mc-#131 >1000.000 — 480.901 —H-A114C-(C114)_MalPeg6C2-#126 54.268 — 656.645 —H-A114C-(C114)_MalPeg6C2-#123 >1000.000 — 543.693 —H-A114C-(C114)_mc-#126 >1000.000 — 749.49 — H-A114C-(C114)_mv-0#1180.147 — 490.276 — H-(C)_MalPeg6C2-#226 0.206 — 582.309 — H-(C)_mc-#2260.219 — 477.622 — H-(C)_m(H2O)c-0#118 0.071 — 306.626 —H-(C)_Mal(H2O)Peg6C2-0#118 <0.059 — 441.766 — H-(C)_Mal(H2O)Peg6C2-#690.203 — 459.502 — H-(C)_m(H2O)c-#69 0.315 — 740.334 — H-(C)_me-0#118<0.061 — 455.314 — H-(C)_mv-0#118 0.084 — 531.617 — H-(C)_mb-0#118 0.076— 584.327 — H-A114C-(C114)_MalC6-#54 52.056 — 65.721 —H-A114C-(C114)_mc-#231 >1000.000 — >1000.000 —H-A114C-(C114)_MalC6-0#118 >1000.000 — >1000.000 —H-(C)_Mal(H2O)Peg6C2-#115 0.095 — 698.101 — H-A114C-(C114)_mc-#158 0.164— 329.554 — H-A114C-(C114)_mcValCitPABC-#231 >1000.000 — >1000.000 —H-(C)_m(H2O)c-#115 <0.069 — 534.743 — H-A114C-(C114)_mc-#237 >1000.000 —646.464 — H-A114C-(C114)_mc-#145 >1000.000 — >1000.000 —H-A114C-(C114)_MalPeg6C2-#145 >1000.000 — >1000.000 —H-A114C-(C114)_mc-#162 0.201 — 676.764 — H-A114C-(C114)_MalC6Am-#1510.469 — 75.696 — H-(kK188)_COPeg2C2ValCitPABC-#54 — — — —H-(C)_mcValCitPABC-0#118 0.081 — >1000.000 —H-A114C-(C114)_mcValCitPABC-#154 1.708 — 566.056 —H-A114C-(C114)_MalC6Am-#153 0.114 — 69.259 — H-(C)_mcValCitPABC-#98 0.23— 270.019 — H-A114C-(C114)_mcValCitPABC-#246 >1000.000 — >1000.000 —H-H435A-(C)_mcValCitPABC-#54 0.208 — 339.77 — H-M428L +N434S-(C)_mcValCitPABC-#70 0.069 — 380.393 — H-M428L +N434S-(C)_mcValCitPABC-#54 0.178 — — — H-E388C + N421C- 1.033 — 826.243— (C388 + C421)_mcValCitPABC-#54 H-Q347C + K392C- 0.103 — 390.7 —(C347 + C392)_mcValCitPABC-#54 H-L443C + kK183C- <0.103 — 395.707 —(C443 + kC183)_mcValCitPABC-#54 H-Q347C + kK183C- <0.051 — 384.028 —(C347 + kC183)_mcValCitPABC-#54 H-Q347C-(C347)_mcValCitPABC-#54 2.89 —393.412 — H-K392C + L443C-(C392 + C443)_mc-#115 0.07 — 542.081 —H-E388C + N421C-(C388 + C421)_mc-#115 0.227 — >1000.000 — H-Q347C +K392C-(C347 + C392)_mc-#115 0.068 — 934.867 — H-L443C + kK183C-(C443 +kC183)_mc- 0.071 — 757.604 — #115 H-Q347C + kK183C-(C347 + kC183)_mc-0.073 — 741.434 — #115 H-Q347C-(C347)_mc-#115 0.098 — 888.128 —H-kK183C-(kC183)_mcValCitPABC-#54 1.329 — 160.012 —H-E388C-(C388)_mcValCitPABC-#54 0.658 — 287.88 —H-kK183C-(kC183)_mc-#115 0.179 — 775.698 — H-E388C-(C388)_mc-#115 0.124— 958.96 — H-L443C-(C443)_mc-#115 0.108 — 451.857 —H-N421C-(C421)_mcValCitPABC-#54 0.601 — 263.107 — H-N421C-(C421)_mc-#1150.108 — 668.857 — H-A114C-(C114)_mcGly-#201 0.073 — — —

TABLE 22 Selected pharmacokinetic values in rats for conjugates of theinvention and selected pharmacokinetic values in rats for conjugatescomprising MMAD, MMAE or MMAF. AUCs were calculated at a 0-last of 0-336h except where noted. AUC (0-last) (μg * Hours/mL) ADC/Ab ADC Dose ADCAb Ratio H(C)-#D54 3   3390¹   4560¹ 74 10  13200¹  16400¹ 80 30  37800² 41700¹ 91 H(C)-#A69 10 14140 20840 68 30 44040 63480 69 100 146000 212000  69 H(C)-MalPEG6C2- 10 13300 15780 84 MMAD 30 56180 60280 93 100134400  146800  92 H(C)-mc-MMAD 10  7650 14500 53 30 20700 43800 47 10058000 121000  48 H(C)-vc-MMAE 3   1080³   2950³ 37 10   3930³  10600³ 3730  13400³  18400³ — H(C)-mc-MMAF 10 10700 24500 44 30 32000 71500 45100 83600 176000  48 H(K)-MCC-DM1 3  3800  5200 73 10 12800 16200 79 3039100 49600 79 ¹denotes a 0-last of 0-312 hours ²denotes a 0-last of0-168 hours ³denotes a 0-last of 0-96 hours

TABLE 23 Selected pharmacokinetic values in mice for conjugates of theinvention and for conjugates comprising MMAD, MMAE or MMAF. AUCs werecalculated at a 0-last of 0-336 h except where noted. AUC (0-last) (μg *Hours/mL) ADC/Ab ADC Dose ADC Ab Ratio H(C)-#D44 3  1070¹  2720¹ 39H(C)-#D70 3 2240 4890 46 H(C)-#D69 3 2490 4770 52 H(C)-#A69 3 3594 572263 H(C)-MalPEG6C2- 3 2641 5415 49 MMAD H(C)-mc-MMAD 3 3580 4970 72H(C)-vc-MMAE 3 1600 3290 49 H(C)-mc-MMAF 3 3080 4800 64 ¹denotes a0-last of 0-168 hours

TABLE 24 Data showing stability of conjugates prepared using ring-openedversus ring closed succinimide-based linkers. GSH stability (6 d) (%loading Mouse ADC remaining AUC Mouse PK Herceptin ADC on day 6) (ug *h/mL) ADC/Ab mc-#118 ring-closed 65% 2160 55% ring-opened 87% 3490 65%MalPeg6C2-#118 ring-closed 82% 2010 70% ring-opened 100%  3000 77%mc-#8261 ring-closed 51% 3590 52% ring-opened 96% 4470 73% MalPeg6C2-ring-closed 61% 2950 72% #8261 ring-opened 104%  — — mc-#115 ring-closed— 1930 58% ring-opened — 2330 68%

TABLE 25A Selected payloads and their methods of synthesis Prepared inthe Same Manner as or Quantity Preparation Purification in mg ExampleMethod Method (Yield) #220 example #107 Method M 10.5 mg (43%) #221example #107 Method M 15.2 mg (76%) #222 General procedure L Method J*14 mg (39%) #223 General procedure L Method J* 16.6 mg (42%) #224General procedure L Method J* 18.8 mg (68%) #225 General procedure LMethod J* 17.3 mg (64%) #226 example #146 silica 354 mg (78%)chromatography #227 General procedure L Method J* 19.4 mg (77%) #228example #131 Method E1* 30 mg (51%) #229 example #151 Method J* 16 mg(61%) #230 General procedure L Method J* 69 mg (42%) #231 Generalprocedure Method J* 4.2 mg (44%) L* #232 example #98 Method J* 113 mg(50%) #233 example #146 silica 88 mg (82%) chromatography #234 Generalprocedure L Method J* 8.5 mg (78%) #235 General procedure Method J* 27mg (77%) L* #236 example #131 achiral 3.7 mg (14%) #237 example #145silica 38.6 mg (93%) chromatography #238 example #145 silica 419 mg(81%) chromatography #239 example #130 silica 315 mg (48%)chromatography #240 example #142 Method E1* 6 mg (20%) #241 example #142Method E1* 6 mg (20%) #242 example #145 Method J* 8 mg (10%) #243example #145 Method J* 12 mg (22%) #244 example #145 Method J* 9.6 mg(20%) #245 General procedure M medium pressue 38 mg (55%) C18 #246example #130 medium pressue 78 mg (80%) C18 #247 example #178 Method M*10.5 mg (57%)

TABLE 25B Selected payloads and their IUPAC name and characterizationdata Mass spectrum: LC-MS or HPLC observed m/z and retention time inminutes: ¹H NMR (400 MHz, Example IUPACNAME DMSO-d₆) unless indicatedotherwise #220 2-methylalanyl-N-[(3R,4S,5S)-1- HPLC (Protocol CB): m/z746.51 [M + H⁺] (1.57 {(2S)-2-[(1R,2R)-3-{[(2R,4S)-4- minutes)carboxy-1-phenylpentan-2- yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3- methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-1-valinamide #221 2-methylalanyl-N-[(3R,4S,5S)-1- HPLC(Protocol DB): m/z 622.42 [M + H⁺] (1.57 {(2S)-2-[(1R,2R)-3- minutes)(bicyclo[1.1.1]pent-1-ylamino)-1- methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3- methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide #222 2-methylalanyl-N-[(3R,4S,5S)-3- LC-MS(Protocol H): m/z 744.9 [M + H⁺] (2.19 minutes).methoxy-1-{(2S)-2-[(1R,2R)-1- ¹H NMR (400 MHz, CDCl₃) δ 7.16-7.22 (m),methoxy-3-{[(1R)-2-methoxy-2- 6.99-7.08 (m), 6.42-6.51 (m), 6.10-6.17(m), 4.87-4.96 (m), oxo-1-(1- 4.65-4.79 (m), 4.27-4.36 (m), 4.04-4.27(m), 3.95-4.02 (m), phenylcyclopropyl)ethyl]amino}- 3.87-3.93 (m),3.64-3.84 (m), 3.44-3.57 (m), 2-methyl-3-oxopropyl]pyrrolidin- 3.22-3.42(m), 3.08-3.17 (m), 2.98-3.07 (m), 2.90-2.93 (m),1-yl}-5-methyl-1-oxoheptan-4-yl]- 2.85-2.89 (m), 2.53-2.57 (m),2.35-2.51 (m), 2.19-2.27 (m), N-methyl- 2.02-2.16 (m), 1.93-2.00 (m),1.77-1.90 (m), L-valinamide 1.57-1.70 (m), 1.35-1.52 (m), 1.26-1.33 (m),1.19-1.25 (m), 1.11-1.16 (m), 1.03-1.11 (m), 0.83-1.02 (m), 0.79-0.88(m). #223 2-methylalanyl-N-[(3R,4S,5S)-3- LC-MS (Protocol H): m/z 744.4[M + H⁺] (2.17 minutes). methoxy-1-{(2S)-2-[(1R,2R)-1- ¹H NMR (400 MHz,CD₃OD) δ 8.19-8.24 (m), methoxy-3-{[(1S)-2-methoxy-2- 7.87-7.92 (m),7.20-7.38 (m), 4.71-5.04 (m), 4.61-4.71 (m), oxo-1-(1- 4.47-4.52 (m),4.38-4.44 (m), 4.05-4.13 (m), phenylcyclopropyl)ethyl]amino}- 3.99-4.04(m), 3.90-3.98 (m), 3.64-3.73 (m), 3.52-3.60 (m),2-methyl-3-oxopropyl]pyrrolidin- 3.46-3.52 (m), 3.37-3.46 (m), 3.35-3.37(m), 3.29-3.35 (m), 1-yl}-5-methyl-1-oxoheptan-4-yl]- 3.24-3.28 (m),3.15-3.19 (m), 3.08-3.14 (m), N-methyl- 3.01-3.06 (m), 2.84-2.87 (m),2.43-2.63 (m), 1.96-2.20 (m), L-valinamide 1.68-1.95 (m), 1.60-1.66 (m),1.52-1.57 (m), 1.33-1.44 (m), 1.27-1.32 (m), 1.23-1.27 (m), 1.12-1.17(m), 1.04-1.10 (m), 0.96-1.03 (m), 0.90-0.96 (m), 0.82-0.90 (m). #2242-methylalanyl-N-[(3R,4S,5S)-1- LC-MS (Protocol H): m/z 730.8 [M + H⁺](2.15 minutes). {(2S)-2-[(1R,2R)-3-({(1R)-1- ¹H NMR (400 MHz, CD₃OD) δ7.09-7.18 (m), [(7R)-bicyclo[4.2.0]octa-1,3,5- 6.95-7.08 (m), 4.88-4.93(m), 4.75-4.85 (m), 4.72-4.74 (m), trien-7-yl]-2-methoxy-2- 4.62-4.70(m), 4.50-4.59 (m), 4.09-4.16 (m), oxoethyl}amino)-1-methoxy-2-3.96-4.06 (m), 3.82-3.90 (m), 3.67-3.76 (m), 3.58-3.67 (m),methyl-3-oxopropyl]pyrrolidin-1- 3.58-3.67 (m), 3.45-3.54 (m), 3.33-3.44(m), 3.33-3.44 (m), yl}-3-methoxy-5-methyl-1-oxoheptan- 3.28-3.33 (m),3.10-3.27 (m), 3.00-3.10 (m), 4-yl]-N-methyl-L- 2.93-3.00 (m), 2.75-2.78(m), 2.56-2.65 (m), 2.36-2.45 (m), valinamide 2.17-2.35 (m), 1.94-2.16(m), 1.67-1.94 (m), 1.48-1.67 (m), 1.27-1.33 (m), 1.23-1.27 (m),1.17-1.26 (m), 1.08-1.17 (m), 0.98-1.07 (m), 0.86-0.98 (m), 0.77-0.84(m). #225 2-methylalanyl-N-[(3R,4S,5S)-1- LC-MS (Protocol H): m/z 730.9[M + H⁺] (2.19 minutes) {(2S)-2-[(1R,2R)-3-({(1S)-1-[(7S)-bicyclo[4.2.0]octa-1,3,5-trien-7- yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan- 4-yl]-N-methyl-L- valinamide #226N,2-dimethylalanyl-N- LC-MS (Protocol Q): m/z 732.4 [M + H⁺] (1.24minutes). [(3R,4S,5S)-3-methoxy-1-{(2S)-2- ¹H NMR δ 8.47-8.53 (m),8.24-8.29 (m), 7.81-7.91 (m), [(1R,2R)-1-methoxy-3-{[(2S)-1- 7.14-7.27(m), 4.54-4.75 (m), 4.44-4.54 (m), methoxy-1-oxo-3-phenylpropan-2-3.94-4.02 (m), 3.72-3.78 (m), 3.61-3.69 (m), 3.28-3.36 (m),yl]amino}-2-methyl-3- 3.14-3.28 (m), 2.99-3.08 (m), 2.81-2.97 (m),2.29-2.57 (m), oxopropyl]pyrrolidin-1-yl}-5- 2.16-2.29 (m), 1.91-2.16(m), 1.60-1.87 (m), methyl-1-oxoheptan-4-yl]-N- 1.35-1.53 (m), 0.99-1.33(m), 0.80-0.99 (m), 0.71-0.80 (m). methyl-L-valinamide #2272-methylalanyl-N-[(3R,4S,5S)-1- LC-MS (Protocol Q): m/z 730.4 [M + H⁺](1.29 minutes) {(2S)-2-[(1R,2R)-3-({(1S)-1-[(7R)-bicyclo[4.2.0]octa-1,3,5- trien-7-yl]-2-methoxy-2-oxoethyl}amino)-1-methoxy-2- methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan- 4-yl]-N-methyl-L- valinamide #228N,N,2-trimethylalanyl-N- HPLC (Protocol A*): m/z 746.5 [M + H⁺] (7.103[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes) [(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2- yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5- methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide #229 N,N,2-trimethylalanyl-N- LC-MS (Protocol Q1): m/z732.3 [M + H⁺] (0.70 [(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes){[(1S)-1-carboxy-2- phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1- yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide #2302-methylalanyl-N-[(3R,4S,5S)-1- HPLC (Protocol G): m/z 730.4 [M + H⁺](1.25 minutes) {(2S)-2-[(1R,2R)-3-{[(R)- carboxy(1-phenylcyclopropyl)methyl]amino}- 1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3- methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide #231 difluoro{2-methylalanyl-N- LC-MS(Protocol Q1): m/z 1020.6 [M + H⁺] (0.83 [(3R,4S,5S)-1-{(2S)-2-minutes). ¹H NMR (400 MHz, CD₃OD) δ [(3R,4R,7S)-7-benzyl-15-{2-[(3,5-8.19-8.23 (m), 7.99-8.07 (m), 7.93-7.98 (m), 7.41-7.45 (m),dimethyl-1H-pyrrol-2-yl- 7.23-7.31 (m), 7.17-7.22 (m), 7.00-7.04 (m),6.32-6.37 (m), kappaN)methylidene]-2H-pyrrol-5- 6.20-6.24 (m), 4.72-4.93(m), 4.61-4.69 (m), yl-kappaN}-4-methyl-5,8,13- 4.05-4.17 (m), 3.88-3.93(m), 3.72-3.81 (m), 3.63-3.70 (m), trioxo-2-oxa-6,9,12- 3.56-3.62 (m),3.48-3.56 (m), 3.25-3.44 (m), 3.16-3.25 (m), triazapentadecan- 3.09-3.14(m), 2.98-3.09 (m), 2.81-2.90 (m), 3-yl]pyrrolidin-1-yl}-3-methoxy-2.54-2.67 (m), 2.39-2.53 (m), 2.09-2.32 (m), 1.75-1.97 (m),5-methyl-1-oxoheptan-4-yl]-N- 1.60-1.69 (m), 1.52-1.59 (m), 1.32-1.44(m), 1.28-1.32 (m), methyl-L-valinamidato}boron 1.16-1.21 (m), 0.98-1.09(m), 0.86-0.98 (m), 0.79-0.90 (m). #232 2-methyl-D-prolyl-N-[(3R,4S,5S)-LC-MS (Protocol Q): m/z 769.3 [M + H⁺] (1.34 minutes).3-methoxy-1-{(2S)-2-[(1R,2R)-1- ¹H NMR δ 9.04-9.17 (m), 8.88-8.94 (m),methoxy-2-methyl-3-oxo-3-{[(1S)- 8.70-8.86 (m), 8.62-8.67 (m), 7.79-7.84(m), 7.76-7.79 (m), 2-phenyl-1-(1,3-thiazol-2- 7.65-7.69 (m), 7.61-7.64(m), 7.20-7.31 (m), 7.12-7.20 (m), yl)ethyl]amino}propyl]pyrrolidin-5.44-5.52 (m), 5.34-5.46 (m), 4.70-4.78 (m),1-yl}-5-methyl-1-oxoheptan-4-yl]- 4.56-4.67 (m), 4.47-4.54 (m),3.94-4.04 (m), 3.76-3.83 (m), N-methyl-L-valinamide 3.52-3.61 (m),3.36-3.52 (m), 3.28-3.35 (m), 3.10-3.27 (m), 2.93-3.08 (m), 2.77-2.80(m), 2.64-2.70 (m), 2.35-2.54 (m), 2.09-2.34 (m), 1.96-2.09 (m),1.54-1.88 (m), 1.38-1.52 (m), 1.18-1.36 (m), 1.03-1.13 (m), 0.81-1.01(m), 0.68-0.81 (m). #233 methyl N-{(2R,3R)-3-[(2S)-1- LC-MS (ProtocolQ): m/z 732.2 [M + H⁺] (1.28 minutes). {(3R,4S,5S)-4-[{N-[(3- ¹H NMR δ8.48-8.53 (m), 8.22-8.28 (m), aminooxetan-3-yl)carbonyl]-L- 7.80-7.92(m), 7.14-7.28 (m), 4.74-4.79 (m), 4.54-4.72 (m),valyl}(methyl)amino]-3-methoxy- 4.43-4.52 (m), 4.24-4.35 (m), 4.07-4.12(m), 3.94-4.02 (m), 5-methylheptanoyl}pyrrolidin-2- 3.72-3.78 (m),3.61-3.69 (m), 3.48-3.58 (m), yl]-3-methoxy-2- 3.40-3.48 (m), 3.11-3.35(m), 2.98-3.11 (m), 2.75-2.97 (m), methylpropanoyl}-L- 2.64-2.69 (m),2.30-2.55 (m), 2.17-2.28 (m), 2.03-2.14 (m), phenylalaninate 1.92-2.02(m), 1.59-1.87 (m), 1.35-1.54 (m), 1.21-1.33 (m), 1.112-1.20 (m),1.00-1.09 (m), 0.70-0.98 (m). #234 2-methylalanyl-N-{(3R,4S,5S)-1- LC-MS(Protocol H): m/z 589.9 [M + H⁺²] (2.29[(2S)-2-{(3R,4R,7S,12S)-7-benzyl- minutes). ¹H NMR (400 MHz, CD₃OD) δ14-[3-chloro-4-(propan-2- 8.55-8.61 (m), 8.40-8.45 (m), 8.34-8.39 (m),8.23-8.28 (m), yloxy)phenyl]-4-methyl-12-[4-(8- 8.14-8.19 (m), 7.84-7.95(m), 7.79-7.84 (m), 7.71-7.77 (m), methylimidazo[1,2-a]pyridin-2-7.61-7.68 (m), 7.46-7.52 (m), 7.34-7.40 (m),yl)benzyl]-5,8,14-trioxo-2,9-dioxa- 7.09-7.27 (m), 7.03-7.09 (m),4.77-4.90 (m), 4.58-4.77 (m), 6,13-diazatetradecan- 4.43-4.55 (m),4.17-4.33 (m), 4.07-4.16 (m), 4.00-4.07 (m), 3-yl}pyrrolidin-1-yl]-3-3.79-3.85 (m), 3.58-3.70 (m), 3.44-3.52 (m),methoxy-5-methyl-1-oxoheptan-4- 3.12-3.40 (m), 2.80-3.12 (m), 2.64-2.71(m), 2.62-2.64 (m), yl}-N-methyl-L-valinamide 2.38-2.47 (m), 2.00-2.33(m), 1.66-2.00 (m), 1.46-1.63 (m), 1.29-1.44 (m), 1.07-1.16 (m),0.91-1.07 (m), 0.79-0.87 (m). #235 2-methylalanyl-N-[(3R,4S,5S)-1- LC-MS(Protocol Q1): m/z 944.3 [M + H⁺] (0.84 {(2S)-2-[(1R,2R)-3-{[(2S)-1-{[4-minutes). ¹NMR (400 MHz, CD₃OD) δ (5-fluoro-1,3-benzothiazol-2-yl)-2-8.54-8.59 (m), 8.29-8.33 (m), 7.87-8.02 (m), 7.80-7.87 (m),methylphenyl]amino}-1-oxo-3- 7.68-7.74 (m), 7.62-7.67 (m), 7.20-7.38(m), 4.98-5.06 (m), phenylpropan-2-yl]amino}-1- 4.84-4.97 (m), 4.66-4.79(m), 4.61-4.66 (m), methoxy-2-methyl-3- 4.13-4.19 (m), 3.98-4.04 (m),3.91-3.96 (m), 3.79-3.85 (m), oxopropyl]pyrrolidin-1-yl}-3- 3.64-3.73(m), 3.38-3.56 (m), 3.34-3.38 (m), 3.28-3.34 (m),methoxy-5-methyl-1-oxoheptan- 3.17-3.27 (m), 3.12-3.16 (m), 3.03-3.11(m), 4-yl]-N-methyl-L-valinamide 2.99-3.03 (m), 2.86-2.87 (m), 2.80-2.82(m), 2.69-2.71 (m), 2.31-2.54 (m), 2.27-2.31 (m), 2.06-2.27 (m),1.88-2.00 (m), 1.74-1.88 (m), 1.64-1.74 (m), 1.59-1.64 (m), 1.50-1.59(m), 1.27-1.48 (m), 1.19-1.26 (m), 1.11-1.16 (m), 1.06-1.11 (m),0.96-1.05 (m), 0.86-0.94 (m), 0.77-0.83 (m). #2361,2-dimethyl-D-prolyl-N- LC-MS (Protocol Q1): m/z 758.3 [M + H⁺] (0.74[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes) [(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2- yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5- methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide #237 N,2-dimethylalanyl-N- LC-MS (Protocol Q1): m/z 771.2[M + H⁺] (0.67 [(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes). ¹H NMR (400MHz, CD₃OD) δ {[(2S)-3-(1H-indol-3-yl)-1- 7.95-7.96 (m), 7.48-7.55 (m),7.45-7.48 (m), 7.26-7.31 (m), methoxy-1-oxopropan-2- 6.94-7.18 (m),5.45-5.49 (m), 5.19-5.22 (m), 5.11-5.17 (m),yl]amino}-1-methoxy-2-methyl-3- 4.97-5.00 (m), 4.78-4.87 (m), 4.68-4.77(m), oxopropyl]pyrrolidin-1-yl}-3- 4.59-4.64 (m), 4.27-4.34 (m),3.99-4.16 (m), 3.84-3.92 (m), methoxy-5-methyl-1-oxoheptan-4- 3.78-3.82(m), 3.62-3.78 (m), 3.49-3.59 (m), 3.41-3.49 (m), yl]-N- 3.20-3.41 (m),2.99-3.20 (m), 2.95-2.98 (m), methyl-L-valinamide 2.82-2.86 (m),2.77-2.79 (m), 2.62-2.68 (m), 2.28-2.49 (m), 2.19-2.27 (m), 1.98-2.16(m), 1.56-1.91 (m), 1.31-1.49 (m), 1.19-1.30 (m), 1.15-1.19 (m),1.06-1.13 (m), 0.88-1.03 (m), 0.79-0.87 (m). #238 N,2-dimethylalanyl-N-LC-MS (Protocol Q1): m/z 758.84 [M + H⁺] (0.71[(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes). ¹H NMR (400 MHz, CD₃OD) δ[(1R,2R)-1-methoxy-2-methyl-3- 7.15-7.32 (m), 5.86-6.00 (m), 5.28-5.40(m), 5.17-5.27 (m), oxo-3-{[(2S)-1-oxo-3-phenyl-1- 4.97-5.04 (m),4.69-4.91 (m), 4.57-4.69 (m), 4.05-4.21 (m),(prop-2-en-1-yloxy)propan-2- 3.91-3.96 (m), 3.79-3.88 (m), 3.71-3.78(m), yl]amino}propyl]pyrrolidin-1-yl}- 3.62-3.70 (m), 3.25-3.56 (m),3.15-3.24 (m), 3.08-3.14 (m), 5-methyl-1-oxoheptan-4-yl]-N- 2.90-3.02(m), 2.79-2.87 (m), 2.42-2.52 (m), 2.20-2.38 (m), methyl-L-valinamide2.12-2.20 (m), 2.03-2.12 (m), 2.00-2.03 (m), 1.71-2.1.96 (m), 1.33-1.70(m), 1.23-1.32 (m), 1.17-1.23 (m), 1.12-1.17 (m), 1.05-1.10 (m),0.94-1.05 (m), 0.82-0.89 (m). #239 2-methyl-L-prolyl-N-[(3R,4S,5S)-LC-MS (Protocol Q): m/z 786.6 [M + H⁺] (1.46 minutes).1-{(2S)-2-[(1R,2R)-3-{[(2S)-1- ¹NMR δ 8.35-8.42 (m), 8.21-8.31 (m),tert-butoxy-1-oxo-3-phenylpropan- 8.14-8.20 (m), 7.15-7.29 (m),4.66-4.76 (m), 4.53-4.65 (m), 2-yl]amino}-1-methoxy-2-methyl- 4.46-4.53(m), 4.32-4.42 (m), 4.07-4.15 (m), 3.96-4.04 (m),3-oxopropyl]pyrrolidin-1-yl}-3- 3.76-3.82 (m), 3.41-3.61 (m), 3.30-3.38(m), methoxy-5-methyl-1-oxoheptan-4- 3.16-3.30 (m), 3.08-3.15 (m)2.99-3.08 (m), 2.92-2.96 (m), yl]-N-methyl- 2.78-2.90 (m), 2.63-2.78(m), 2.37-2.58 (m), 2.18-2.36 (m), L-valinamide 2.03-2.13 (m), 1.89-2.01(m), 1.64-1.88 (m), 1.35-1.62 (m), 1.31-1.35 (m), 1.17-1.31 (m),1.03-1.14 (m), 0.70-1.01 (m). #240 N,2-dimethylalanyl-N- LC-MS (ProtocolQ1): m/z 798.2 [M + H⁺] (0.66 [(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes).¹H NMR (400 MHz, CD₃OD) δ [(1R,2R)-1-methoxy-2-methyl-3- 8.43-8.49 (m),7.50-7.53 (m), 7.42-7.48 (m), 7.06-7.20 (m),oxo-3-({(2S)-1-oxo-3-phenyl-1- 4.21-4.83 (m), 3.95-4.13 (m), 3.76-3.88(m), 3.53-3.67 (m), [(1H-1,2,3-triazol-4- 3.16-3.47 (m), 3.08-3.15 (m)3.00-3.16 (m), ylmethyl)amino]propan-2- 2.77-2.90 (m), 2.70-2.73 (m),2.62-2.69 (m), 2.45-2.58 (m), yl}amino)propyl]pyrrolidin-1-yl}-2.34-2.41 (m), 2.21-2.29 (m), 2.12-2.21 (m), 1.55-2.09 (m), 5-methyl-1.39-1.54 (m), 1.16-1.36 (m), 1.04-1.14 (m),1-oxoheptan-4-yl]-N-methyl-L- 0.85-0.99 (m), 0.73-0.80 (m), 0.00-0.02(m). valinamide #241 N,2-dimethylalanyl-N- LC-MS (Protocol Q1): m/z755.1 [M + H⁺] (0.69 [(3R,4S,5S)-3-methoxy-1-{(2S)-2- minutes). ¹H NMR(400 MHz, CD₃OD) δ [(1R,2R)-1-methoxy-2-methyl-3- 8.36-8.67 (m),7.26-7.50 (m), 7.10-7.26 (m), 5.13-5.17 (m),oxo-3-{[(2S)-1-oxo-3-phenyl-1- 4.95-4.99 (m), 4.67-4.84 (m), 4.61-4.66(m), 4.50-4.60 (m), (prop-2-yn-1-ylamino)propan-2- 3.77-4.12 (m),3.69-3.75 (m), 3.56-3.66 (m), yl]amino}propyl]pyrrolidin-1-yl}-3.44-3.54 (m), 3.19-3.44 (m) 3.12-3.19 (m), 3.03-3.12 (m),5-methyl-1-oxoheptan-4-yl]- 2.74-2.94 (m), 2.37-2.60 (m), 2.14-2.36 (m),1.60-2.13 (m), N-methyl-L-valinamide 1.47-1.59 (m), 1.19-1.40 (m),1.11-1.16 (m), 0.88-1.11 (m), 0.75-0.84 (m), 0.02-0.06 (m). #242N,2-dimethylalanyl-N- LC-MS (Protocol Q1): m/z 722.95 [M + H⁺] (0.52[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes) ¹H NMR (400 MHz, CD₃OD) δ8.78-8.86 (m), {[(2S)-3-(1H-imidazol-4-yl)-1- 8.71-8.73 (m), 7.96-8.00(m), 7.34-7.40 (m), methoxy-1-oxopropan-2- 4.74-4.91 (m), 4.67-4.71 (m),4.55-4.63 (m), 4.13-4.22 (m), yl]amino}-1-methoxy-2-methyl-3- 4.04-4.10(m), 3.97-4.01 (m), 3.84-3.92 (m), 3.66-3.82 (m),oxopropyl]pyrrolidin-1-yl}-3- 3.42-3.64 (m), 3.26-3.42 (m) 3.11-3.21(m), methoxy-5-methyl-1-oxoheptan-4- 2.90-2.92 (m), 2.83-2.84 (m),2.59-2.64 (m), 2.48-2.56 (m), yl]- 2.32-2.41 (m), 2.09-2.24 (m),1.99-2.08 (m), 1.68-1.95 (m), N-methyl-L-valinamide 1.59-1.66 (m),1.51-1.58 (m), 1.35-1.45 (m). 1.22-1.26 (m), 1.17-1.21 (m). 0.95-1.12(m), 0.83-0.89 (m). #243 N,2-dimethylalanyl-N- LC-MS (Protocol Q1): m/z748.2 [M + H⁺] (0.52 [(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes) ¹H NMR(400 MHz, CD₃OD), δ 8.91-8.99 (m), {[(2S)-3-(4-hydroxyphenyl)-1-8.42-8.46 (m), 8.15-8.20 (m), 7.92-8.01 (m), methoxy-1-oxopropan-2-7.00-7.10 (m), 6.64-6.74 (m), 5.22-5.26 (m), 5.06-5.09 (m),yl]amino}-1-methoxy-2-methyl-3- 4.79-4.95 (m), 4.65-4.79 (m), 4.59-4.65(m), 4.12-4.21 (m), oxopropyl]pyrrolidin-1-yl}-3- 4.05-4.12 (m),3.91-3.99 (m), 3.84-3.90 (m), methoxy-5-methyl-1-oxoheptan-4- 3.67-3.79(m), 3.60-3.66 (m), 3.39-3.57 (m), 3.34-3.39 (m) yl]- 3.29-3.34 (m),3.12-3.27 (m), 2.98-3.00 (m), 2.78-2.88 (m), N-methyl-L-valinamide2.61-2.65 (m), 2.55-2.57 (m), 2.46-2.53 (m), 2.10-2.36 (m), 1.68-1.96(m), 1.61-1.68 (m), 1.55-1.60 (m), 1.35-1.53 (m), 1.19-1.24 (m),1.14-1.18 (m), 1.08-1.13 (m), 0.98-1.08 (m), 0.84-0.92 (m). #244N,2-dimethylalanyl-N- LC-MS (Protocol Q1): m/z 718.4 [M + H⁺] (0.66[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- minutes) ¹H NMR (400 MHz, CD₃OD), δ{[(1R)-1-carboxy-2- 7.87-7.92 (m), 7.71-7.76 (m), 7.46-7.53 (m),7.40-7.46 (m), phenylethyl]amino}-1-methoxy-2- 7.19-7.33 (m), 4.81-4.96(m), 4.68-4.77 (m), 4.60-4.65 (m), methyl-3-oxopropyl]pyrrolidin-1-4.47-4.53 (m), 4.01-4.17 (m), 3.94-3.98 (m), yl}-3-methoxy-5-methyl-1-3.81-3.86 (m), 3.68-3.76 (m), 3.56-3.64 (m), 3.40-3.50 (m),oxoheptan-4-yl]-N-methyl-L- 3.36-3.40 (m) 3.26-3.35 (m), 3.23-3.26 (m),3.16-3.22 (m), valinamide 3.12-3.16 (m), 2.94-3.06 (m), 2.91-2.93 (m),2.86-2.88 (m), 2.41-2.66 (m), 2.32-2.41 (m), 1.97-2.23 (m), 1.85-1.97(m), 1.71-1.85 (m), 1.62-1.68 (m), 1.50-1.61 (m), 1.37-1.46 (m),0.98-1.14 (m), 0.85-0.92 (m). #245 1,2-dimethyl-L-prolyl-N- LC-MS(Protocol Q): m/z 835.0 [M + Na⁺] (0.87 [(3R,4S,5S)-3-methoxy-1-{(2S)-2-minutes) ¹H NMR δ 9.58-9.69 (m), 8.84-9.16 (m),[(1R,2R)-1-methoxy-2-methyl-3- 8.69-8.77 (m), 8.54-8.60 (m), 8.44-8.50(m), 8.32-8.42 (m), oxo-3-{[(2S)-1-oxo-3-phenyl-1- 8.25-8.30 (m),7.13-7.31 (m), 7.00-7.01 (m), (piperazin-1-yl)propan-2- 4.97-5.06 (m),4.88-4.97 (m), 4.57-4.75 (m), 4.45-4.57 (m),yl]amino}propyl]pyrrolidin-1-yl}- 3.84-4.45 (m), 3.62-3.84 (m),3.40-3.62 (m), 3.13-3.33 (m), 5-methyl-1-oxoheptan-4-yl]- 2.77-3.10 (m),2.67-2.75 (m), 2.47-2.57 (m), N-methyl-L-valinamide 2.38-2.45 (m),1.92-2.35 (m), 1.58-1.88 (m), 1.37-1.55 (m), 1.22-1.32 (m), 0.97-1.06(m), 0.84-0.97 (m), 0.73-0.81 (m). #246 1,2-dimethyl-L-prolyl-N- LC-MS(Protocol Q): m/z 366.2 [M + H⁺²] (0.91 minutes)[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- ¹H NMR δ 9.56-9.65 (m), 8.70-8.76 (m),8.05-8.09 (m), {[(2S)-1-amino-3-phenylpropan-2- 7.77-7.92 (m), 7.14-7.30(m), 4.60-4.72 (m), yl]amino}-1-methoxy-2-methyl-3- 4.46-4.57 (m),3.61-4.39 (m), 3.41-3.61 (m), 3.11-3.33 (m),oxopropyl]pyrrolidin-1-yl}-3- 2.97-3.09 (m), 2.79-2.94 (m), 2.63-2.74(m), 2.38-2.56 (m), methoxy-5-methyl-1-oxoheptan-4- 2.13-2.37 (m),1.93-2.13 (m), 1.45-1.89 (m), yl]-N-methyl-L-valinamide 1.21-1.32 (m),1.09-1.14 (m), 1.03-1.08 (m), 0.84-095 (m), 0.73-0.80 (m). #2472-methyl-D-prolyl-N-[(3R,4S,5S)- HPLC (Protocol DB): m/z 700.51 [M + H⁺](2.56 1-{(2S)-2-[(1R,2R)-3-{[2- minutes) (cyclohepta-2,4,6-trien-1-yl)ethyl]amino}-1-methoxy-2- methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1- oxoheptan-4-yl]-N-methyl-L-valinamide

We claim:
 1. A compound of formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl; R² is hydrogen, C₁-C₈alkyl or C₁-C₈ haloalkyl; R^(3A) and R^(3B) are either of the following:(i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; and R^(3B)is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl,aryl, heteroaralkyl, aralkyl or halogen; or (ii) R^(3A) and R^(3B) takentogether are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R^(4A) and R^(4B)are either of the following: (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkylor aralkyl; and R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; or(ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or C₁-C₈heteroalkylene; R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; or R⁵is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; R⁶is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl; R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄aryl; R¹³ is C₁-C₁₀ heterocyclyl; and X is O or S; provided that whenR^(3A) is hydrogen X is S.
 2. A compound of formula IIa:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, W is

R¹ is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-; —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-or —C₃-C₈ heterocyclo)-C₁-C₁₀alkylene-; Z is

G is halogen, —OH, —SH or —S—C₁-C₆ alkyl; R² is hydrogen, C₁-C₈ alkyl orC₁-C₈ haloalkyl; R^(3A) and R^(3B) are either of the following: (i)R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R^(3B)is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl,aryl, heteroaralkyl or aralkyl or halogen; or (ii) R^(3A) and R^(3B)taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R^(4A) andR^(4B) are either of the following: (i) R^(4A) is hydrogen, C₁-C₈ alkyl,C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl,heteroaralkyl or aralkyl; and R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkylor aralkyl; or (ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkyleneor C₁-C₈ heteroalkylene; R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; or R⁵is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —O(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; R⁶is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl; R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄aryl; R¹³ is C₁-C₁₀ heterocyclyl; and R⁷ is independently selected foreach occurrence from the group consisting of F, Cl, I, Br, NO₂, CN andCF₃; R¹⁰ is hydrogen, —C₁-C₁₀alkyl, —C₃-C₈-carbocyclyl, -aryl,—C₁-C₁₀heteroalkyl, —C₃-C₈heterocyclo, —C₁-C₁₀alkylene-aryl,-arylene—C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo), —(C₃-C₈carbocyclo)-C₁-C₁₀alkyl, —C₁-C₁₀alkylene-(C₃-C₈heterocyclo), and —(C₃-C₈heterocyclo)-C₁-C₁₀alkyl, where aryl on R₁₀ comprising aryl isoptionally substituted with [R₇]_(h); h is 1, 2, 3, 4 or 5; and X is Oor S; provided that when R^(3A) is hydrogen X is S.
 3. A compound offormula IIIa:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl; R² is hydrogen, C₁-C₈alkyl or C₁-C₈ haloalkyl; R^(3A) and R^(3B) are either of the following:(i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R^(3B)is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl,aryl, heteroaralkyl, halogen or aralkyl; or (ii) R^(3A) and R^(3B) takentogether are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R^(4A) and R^(4B)are either of the following: (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkylor aralkyl; and R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; or(ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or C₁-C₈heteroalkylene; R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R)₂, —NHC(O)R′,—S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂, —CN,—NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl; R¹¹is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈ heterocyclo-, —C₁-C₁₀alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁alkylene-(C₃-C₁₀-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₁₀heterocyclo)-, or -(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-; Z is

R⁷ is independently selected for each occurrence from the groupconsisting of F, Cl, I, Br, NO₂, CN and CF₃; R¹⁰ is hydrogen,—C₁-C₁₀alkyl, —C₃-C₈-carbocycle, aryl, —C₁-C₁₀ heteroalkyl,—C₃-C₈heterocyclo, —C₁-C₁₀alkylene-aryl, -arylene-C₁-C₁₀alkyl,—C₁-C₁₀alkylene-(C₃-C₁₀-carbocyclo), —(C₃-C₁₀ carbocyclo)-C₁-C₁₀alkyl,—C₁-C₁₀alkylene-(C₃-C₁₀heterocyclo), and -(C₃-C₁₀heterocyclo)-C₁-C₁₀alkyl, where aryl on R₁₀ comprising aryl isoptionally substituted with [R₇]_(h); X is O or S R⁶ is hydrogen, C₁-C₈alkyl or C₁-C₈ haloalkyl; and h is 1, 2, 3, 4 or
 5. 4. A compound offormula IIb:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, W is

R¹ is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-; Z is

L is an antibody; R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl; R^(3A)and R^(3B) are either of the following: (i) R^(3A) is hydrogen, C₁-C₈alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl,heteroaralkyl, aralkyl or halogen; and R^(3B) is C₁-C₈ alkyl, C₁-C₈haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl,halogen or aralkyl; or (ii) R^(3A) and R^(3B) taken together are C₂-C₈alkylene or C₁-C₈ heteroalkylene; R^(4A) and R^(4B) are either of thefollowing: (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; andR^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-O₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R^(4A) andR^(4B) taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; or R⁵is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl, C₁-C₁₀alkylene—C₃-C₈heterocyclyl and aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; R⁶ ishydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl; R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄aryl; R¹³ is C₁-C₁₀ heterocyclyl; and X is O or S; provided that whenR^(3A) is hydrogen X is S.
 5. A compound of formula IIIb:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, W is R¹ is hydrogen, C₁-C₈ alkyl orC₁-C₈ haloalkyl;

R² is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl; R^(3A) and R^(3B) areeither of the following: (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl,aralkyl or halogen; and R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen oraralkyl; or (ii) R^(3A) and R^(3B) taken together are C₂-C₈ alkylene orC₁-C₈ heteroalkylene; R^(4A) and R^(4B) are either of the following: (i)R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(4B) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R^(4A) and R^(4B)taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R)₂, —NHC(O)R′,—S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂, —CN,—NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl; R¹¹ is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-; Z is

L is an antibody; X is O or S.
 6. A compound of formula IIc:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, R^(1′) is

Y is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-; Z′ is

L is an antibody; D is —C(R^(4A′))(R^(4B′))— or is absent; R^(2′) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, or is absent if

is present; R^(3A′) and R^(3B′) are either of the following: (i) R^(3A′)is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R^(3B′) isC₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl,aryl, heteroaralkyl, halogen or aralkyl, or R^(3B′) is C₂-C₄ alkyleneand forms 5-7 member ring as indicated by

; or (ii) R^(3A′) and R^(3B′) taken together are C₂-C₈ alkylene or C₁-C₈heteroalkylene; R^(4A′) and R^(4B′) are either of the following: (i)R^(4A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(4B′) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R^(4A′) andR^(4B′) taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; or R⁵is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl, C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; R⁶ ishydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl; R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄aryl; R¹³ is C₁-C₁₀ heterocyclyl; and X is O or S; provided that whenR^(3A) is hydrogen X is S.
 7. A compound of formula IIIc:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence,

W is R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl; R² is hydrogen,C₁-C₈ alkyl or C₁-C₈ haloalkyl; R^(3A) and R^(3B) are either of thefollowing: (i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl orhalogen; and R^(3B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl; or (ii)R^(3A) and R^(3B) taken together are C₂-C₈ alkylene or C₁-C₈heteroalkylene; R^(4A) and R^(4B) are either of the following: (i)R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(4B) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R^(4A) and R^(4B)taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R)₂, —NHC(O)R′,—S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂, —CN,—NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl;

Y is is —C₂-C₂₀ alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-,-arylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-arylene-,-arylene-C₁-C₁₀alkylene-, —C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-; Z′ is

L is an antibody; X is O or S.
 8. A compound of formula IId:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, L is an antibody; [linker] is adivalent linker; D is —C(R^(4A′))(R^(4B′))— or is absent; R^(2′) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, or is absent if

, is present; R^(3A′) and R^(B′) are either of the following: (i)R^(3A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; andR^(3B′) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl, halogen or aralkyl, or R^(3B′) isC₂-C₄ alkylene and forms 5-7 member ring as indicated by

; or (ii) R^(3A′) and R^(3B′) taken together are C₂-C₈ alkylene or C₁-C₈heteroalkylene; R^(4A′) and R^(4B′) are either of the following: (i)R^(4A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(4B′) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; or (ii) R^(4A′) andR^(4B′) taken together are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R⁵ is

C₁-C₁₀ heterocyclyl, C₃-C₈ carbocycly and C₆-C₁₄ aryl optionallysubstituted with 1, 2, 3, 4 or 5 groups independently selected from thegroup consisting of —C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂, —C₁-C₈alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′—O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, halogen, —N₃,—N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein eachR′ is independently selected from the group consisting of hydrogen,C₁-C₈ alkyl and unsubstituted aryl, or two R′ can, together with thenitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; or R⁵is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —C₁-C₈ alkyl-N(R′)₂,—C₁-C₈ alkyl-C(O)R′, —C₁-C₈ alkyl-C(O)OR′, —O—(C₁-C₈ alkyl), —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,halogen, —N₃, —N(R′)₂, —CN, —NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′, —SR′ andarylene-R′, wherein each R′ is independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl, C₁-C₈heterocyclyl,C₁-C₁₀alkylene-C₃-C₈heterocyclyl and aryl, or two R′ can, together withthe nitrogen to which they are attached, form a C₁-C₁₀ heterocyclyl; R⁶is hydrogen, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl or —C₁-C₈haloalkyl; R¹² is hydrogen, C₁-C₄ alkyl, C₁-C₁₀ heterocyclyl or C₆-C₁₄aryl; R¹³ is C₁-C₁₀ heterocyclyl; and X is O or S; provided that whenR^(3A) is hydrogen X is S.
 9. A compound of formula IIId:

or a pharmaceutically acceptable salt or solvate thereof, wherein,independently for each occurrence, W is

R¹ is hydrogen, C₁-C₈ alkyl or C₁-C₈ haloalkyl; R² is hydrogen, C₁-C₈alkyl or C₁-C₈ haloalkyl; R^(3A) and R^(3B) are either of the following:(i) R^(3A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl,C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and R^(3B)is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl,aryl, heteroaralkyl, halogen or aralkyl; or (ii) R^(3A) and R^(3B) takentogether are C₂-C₈ alkylene or C₁-C₈ heteroalkylene; R^(4A) and R^(4B)are either of the following: (i) R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkylor aralkyl; and R^(4B) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; or(ii) R^(4A) and R^(4B) taken together are C₂-C₈ alkylene or C₁-C₈heteroalkylene; R⁵ is

optionally substituted with 1, 2, 3, 4 or 5 groups independentlyselected from the group consisting of C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),—C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R)₂, —NHC(O)R′,—S(O)₂R′, —S(O)R′, —OH, halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂, —CN,—NHC(═NH)NH₂, —NHCONH₂, —S(═O)₂R′ and —SR′, wherein each R′ isindependently selected from the group consisting of hydrogen, C₁-C₈alkyl and unsubstituted aryl; [linker] is a divalent linker; L is anantibody; X is O or S.
 10. The compound, salt or solvate of claim 1,wherein W is


11. The compound, salt or solvate of claim 1, wherein R¹ is hydrogen,C₁-C₈ alkyl or C₁-C₈ haloalkyl.
 12. The compound, salt or solvate ofclaim 2, wherein R¹ is


13. The compound, salt or solvate of claim 4 wherein R¹ is


14. The compound, salt or solvate of claim 1, wherein R^(3A) ishydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(3B) is C₁-C₈ alkyl,C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl,heteroaralkyl, aralkyl or halogen.
 15. The compound, salt or solvate ofclaim 1, wherein R^(3A) and R^(3B) taken together are C₂-C₈ alkylene orC₁-C₈ heteroalkylene.
 16. The compound, salt or solvate of claim 1,wherein R^(4A) is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl or aralkyl; andR^(4B) is C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; or R^(4A) and R^(4B) takentogether are C₂-C₈ alkylene or C₁-C₈ heteroalkylene.
 17. The compound,salt or solvate of claim 6, wherein R^(3A′) and R^(3B′) are defined aseither of the following: (i) R^(3A′) is hydrogen, C₁-C₈ alkyl, C₁-C₈haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl,aralkyl or halogen; and R^(3B′) C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈carbocyclyl, C₁-C₁₀ heterocyclyl, aryl, heteroaralkyl, halogen oraralkyl, or R^(3B′) is C₂-C₄ alkylene and forms 5-7 member ring asindicated by

; or (ii) R^(3A′) and R^(3B′) taken together are C₂-C₈ alkylene or C₁-C₈heteroalkylene.
 18. The compound, salt or solvate of claim 6, whereinR^(4A′) and R^(4B′) are defined as either of the following: (i) R^(4A′)is hydrogen, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀heterocyclyl, aryl, heteroaralkyl or aralkyl; and R^(4B′) is hydrogen,C₁-C₈ alkyl, C₁-C₈ haloalkyl, C₃-C₈ carbocyclyl, C₁-C₁₀ heterocyclyl,aryl, heteroaralkyl or aralkyl; or (ii) R^(4A′) and R^(4B′) takentogether are C₂-C₈ alkylene or C₁-C₈ heteroalkylene.
 19. The compound,salt or solvate of claim 1, wherein R⁵ is


20. The compound, salt or solvate of claim 2, wherein Y is —C₂-C₂₀alkylene-, —C₂-C₂₀ heteroalkylene-, —C₃-C₈ carbocyclo-, -arylene-,—C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈-carbocyclo)-,—(C₃-C₈-carbocyclo)-C₁-C₁₀alkylene-,—C₁-C₁₀alkylene-(C₃-C₈heterocyclo)-, or —(C₃-C₈heterocyclo)-C₁-C₁₀alkylene-.
 21. The compound, salt or solvate of claim5, wherein antibody is selected from trastuzumab, oregovomab,edrecolomab, cetuximab, a humanized monoclonal antibody to thevitronectin receptor (α_(v)β₃); alemtuzumab; a humanized anti-HLA-DRantibody for the treatment of non-Hodgkin's lymphoma; 131I Lym-1; amurine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin'slymphoma; a humanized anti-CD2 mAb for the treatment of Hodgkin'sDisease or non-Hodgkin's lymphoma; labetuzumab; bevacizumab; ibritumomabtiuxetan; ofatumumab; panitumumab; rituximab; tositumomab; ipilimumab;gemtuzumab; an anti-IL13 antibody; and an anti-Notch antibody.
 22. Acompound, or a pharmaceutically acceptable salt or solvate thereof,selected from:


23. A compound, or a pharmaceutically acceptable salt or solvatethereof, selected from:


24. A payload-linker comprising a radical of a compound of claim 22 or23.
 25. An antibody drug conjugate comprising a radical of a compound ofany of claims 1, 2, or
 3. 26. A compound, or a pharmaceuticallyacceptable salt or solvate thereof, selected from:


27. An antibody-drug-conjugate comprising a radical of a compound of anyone of claims 22, 23, 24, 25, or
 26. 28. A pharmaceutical compositioncomprising a compound of claim 1, or a pharmaceutically acceptable saltor solvate thereof, and pharmaceutically acceptable excipient.
 29. Amethod of treating cancer comprising administering to a patient in needthereof a therapeutically effective amount of a compound of claim
 4. 30.The compound:

or a pharmaceutically acceptable salt or solvate thereof.
 31. Thecompound:

or a pharmaceutically acceptable salt or solvate thereof.
 32. Thecompound:

or a pharmaceutically acceptable salt or solvate thereof.
 33. Thecompound:

or a pharmaceutically acceptable salt or solvate thereof.
 34. Thecompound:

or a pharmaceutically acceptable salt or solvate thereof.
 35. Thecompound:

or a pharmaceutically acceptable salt or solvate thereof.
 36. Thecompound:

or a pharmaceutically acceptable salt or solvate thereof.